An automated multi-functional BIO-analyser

Abstract

The present invention relates to an automated integrated system for detecting nucleic acid, protein, antigen and antibodies in a given sample, using Polymerase Chain Reaction (PCR), Time-Resolved Fluorescence Immunoassay (TRFIA) and Chemiluminescent Immunoassay (CLIA) techniques. The system (100) comprises a portable analyser (103) for automated sample movement, incubation, thermal cycling, and optical readouts; and a single-use disposable cartridge (102) for sample processing. The said cartridge (102) is prefilled with all necessary reagents and buffers to streamline workflow and minimize user intervention. Further, the samples can include blood, body fluids, animal, human, plant or food matrix, environmental samples, and forensic sample. The said automated multifunctional bio-analyser (100) facilitates reduced manual intervention, increased throughput, and improved accuracy and reliability of diagnostic assays, making it suitable for use in clinical laboratories, research settings, and field diagnostics.

Description

[0001] AN AUTOMATED MULTI-FUNCTIONAL BIO-ANALYSER FIELD OF INVENTION:

[0002]

[0001] The present invention generally relates to a healthcare diagnostic device. More particularly, the present invention relates to an automated integrated system capable of detecting nucleic acid, proteins, antigens and antibodies in a given sample, using Polymerase Chain Reaction (PCR), Time-Resolved Fluorescence Immunoassay (TRFIA) and Chemiluminescent Immunoassay (CLIA) techniques.

[0003] BACKGROUND AND PRIOR ART:

[0004]

[0002] Traditional diagnostic assays typically rely on single technique for detecting and quantifying biological targets. Polymerase chain reaction (PCR) is widely used for amplification of nucleic acids, while time-resolved fluorescence immunoassay (TRFIA) and chemiluminescent immunoassay (CLIA) are widely used for identifying proteins or other analytes by fluorescence and chemiluminescence, respectively. However, integrating these techniques in a single system has proven challenging due to issues related to accuracy, efficiency, and operating complexity. The existing systems often operate these assays separately, limiting the ability to detect multiple analytes and achieving high sensitivity and specificity.

[0005]

[0003] Further, decentralisation of laboratory testing is very much essential for fast and timely detection of various disease conditions, especially in low resource settings where the accessibility of advanced laboratory support is time taking.

[0006]

[0004] Moreover, there is also an increasing demand for a user-friendly and efficient diagnostic system that can perform multimodal analysis quickly and with minimal sample processing. A unified system which can directly accept the clinical or biological samples, and can conduct real-time PCR, TRFIA and CLIAwould be of great use for analysing numerous clinical cases, enabling prompt diagnosis and informed clinical decision making.

[0007]

[0005] Currently, samples for PCR, TRFIA and CLIA are processed separately and require different machines.

[0008]

[0006] RU2017131209A discloses a device and method for observing, testing and analyzing biological samples. The said system comprises: a sample preparation unit configured to receive a sample holder having a plurality of samples; a control system configured to cycle multiple samples at various temperatures; and an automated tray with movable assembly to enable reversible sliding movement between the closed and open positions to provide the user with access to the plurality of sample holders. Further, said tray or assembly comprises a plate configured to block light emitted from the position sensor. However, the system according to RU’209 has increased complexity which results in reduced effectiveness.

[0009]

[0007] Thus, there is a need for a unified system that can detect nucleic acids, proteins, antigens, and antibodies within a single machine to enhance diagnostic capabilities efficiently and effectively.

[0010]

[0008] The present invention aims to provide an automated integrated diagnostic system capable of performing nucleic acid extraction, multiplex real-time polymerase chain reaction (PCR) analysis, time-resolved fluorescence immunoassay (TRFIA), and particle-based chemiluminescent immunoassay (CLIA), thereby offering a comprehensive diagnostic solution from sample collection to final diagnostic output.

[0011] SUMMARY OF THE INVENTION:

[0012]

[0009] The present invention provides an automated multifunctional bio-analysis system for efficient nucleic acid extraction, multiplex real-time polymerase chainreaction (PCR) analysis, time-resolved fluorescence immunoassay (TRFIA), and particle-based chemiluminescent immunoassay (CLIA), using a single analyser platform and assay-specific disposable cartridges, thereby providing an integrated solution from sample collection to final diagnostic output. The system is configured to reduce manual intervention, increase throughput, and improve the accuracy and reliability of diagnostic assay.

[0013]

[0010] The system comprises a single automated analyser configured to receive a plurality of assay-specific disposable cartridges, wherein each cartridge is preconfigured with reagents and buffers suitable for a respective diagnostic technique selected from the group consisting of PCR, TRFIA, or CLIA. Although the cartridges differ in reagent composition and assay workflow, each cartridge includes a standardized mechanical, pneumatic, and optical interface compatible with the analyser, thereby enabling processing of different assay types within the same analyser platform. This configuration permits execution of multiple diagnostic modalities on a common platform while maintaining assay specificity and analytical performance.

[0014] [Oil] It is an object of the present invention to provide a unified diagnostic system that seamlessly integrates PCR, TRFIA, and CLIA diagnosing techniques, to facilitate efficient and automated analysis of biological samples.

[0015]

[0012] It is another object of the present invention to provide a multifunctional system capable of performing fluorescent-based, colorimetric, chemiluminescent analysis in PCR, CLIA and TRFIA diagnosing techniques.

[0016]

[0013] It is yet another object of the present invention to enable amplification and detection of nucleic acids, proteins, antigens and antibodies within a single automated platform, thereby enhancing diagnostic performance.

[0014] It is yet another object of the present invention to achieve increased sensitivity, reduced assay time, and the capability to perform multiplex assays.

[0017]

[0015] It is yet another object of the present invention to enhance the efficiency and reliability of nucleic acid extraction and real-time clinical analysis using PCR, TRFIA and particle-based CLIA.

[0018]

[0016] It is yet another object of the present invention to provide an automated system that reduces manual intervention by ensuring seamless integration of processes from sample preparation to final analysis, thereby preventing contamination, eliminating the need for extensive sample preprocessing, and simplifying user operation.

[0019]

[0017] It is yet another object of the present invention to provide a portable analyzer capable of functioning as a standalone platform in remote laboratory settings and equipped with battery backup for uninterrupted operation.

[0020]

[0018] Accordingly, the present invention discloses an automated multifunctional bio-analyser comprising a portable analyser configured for automated sample movement, incubation, thermal cycling, and optical readouts; and a single-use disposable cartridge for sample processing. The said cartridge is prefilled with reagents and buffers to streamline workflow and minimize user intervention. The biological sample may include blood, body fluids, animal samples, human samples, plant or food matrices, environmental samples, or forensic samples.

[0021]

[0019] The analyzer further comprises process modules including a barcode module, a thermal control module for temperature regulation, a motor module for valve actuation, a fluorescence detection module, a sonicator module, an integrated detection unit and a unified control interface, wherein the modules are operatively connected to a program controller and a data processing and analysis module, to ensure precise and automated operation.

[0020] The system is further configured to perform multiplex detection, wherein multiple nucleic acid targets and / or multiple analytes are simultaneously detected using spatially separated reaction wells and / or spectrally distinguishable optical labels.

[0022]

[0021] The present invention further discloses a method for analysing a biological sample to detect nucleic acids, proteins, antigens and antibodies using real-time PCR, TRFIA and CLIA within said multifunctional bio-analyser system.

[0023] BRIEF DESCRIPTION OF DRAWINGS:

[0024]

[0022] The figures below show an exemplary embodiment:

[0025] Figure 1 illustrates a schematic representation of the cartridge with valves.

[0026] Figure 2A, 2B, illustrates a schematic representation of the cartridge without valves.

[0027] Figure 3 illustrates a schematic representation of the typical channel splitting at the PCR well end in the cartridge.

[0028] Figure 4 illustrates a schematic representation of the cartridge spring holder which covers the port for liquid filling.

[0029] Figure 5A, 5B illustrates a schematic representation of the air chamber for giving a positive pressure to reagent chambers.

[0030] Figure 6 illustrates a schematic representation of the transparent PCR well.

[0031] Figure 7 illustrates a schematic representation of the retaining block fixed on the cartridge top.

[0032] Figure 8 illustrates a schematic representation of the flow control valve 1 and 2.

[0033] Figure 9 illustrates a schematic representation of the flow control valve 3.

[0034] Figure 10 illustrates a schematic representation of the automated multifunctional bio-analyser.

[0035] Figure 11 illustrates a schematic representation of the cartridge carrier mechanism.Figure 12, 12A, 13 illustrates a schematic representation of the various parts of automated multifunctional bio-analyser in detail.

[0036] Figure 14 illustrates a schematic representation of the optic module assembly. Figure 15 illustrates a schematic representation of the thermal block assembly forPCR.

[0037] Figure 16 illustrates a functional block diagram of the automated multifunctional bio-analyser.

[0038] Figure 17 illustrates the optic fiber arrangement for the imaging system.

[0039] Figure 18 illustrates the optic module arrangement for the LED and photodetector unit.

[0040] Figure 19 illustrates a graphical representation of relative fluorescence units (RFU) plotted against PCR cycle number for a four-well configuration, demonstrating comparative amplification performance of the proposed system with a reference system.

[0041] DETAILED DESCRIPTION OF THE INVENTION:

[0042]

[0023] The present invention will now be described in detail with reference to optional and preferred embodiments so that various aspects of the invention will be more clearly understood, however, should not be construed to limit the scope of the invention. The following embodiments clearly and completely describes various technical features and advantageous of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. The examples used herein are intended merely to facilitate an understanding of the ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0043]

[0024] The present invention provides an automated multifunctional bio-analysis system (100) configured to seamlessly integrate polymerase chain reaction (PCR), time-resolved fluorescence immunoassay (TRFIA), and chemiluminescentimmunoassay (CLIA) diagnosing techniques for automated and efficient analysis of nucleic acids, proteins, antigens, and antibodies from biological samples (101). The system (100) provides a comprehensive solution from sample processing to diagnostic output, thereby enhancing analytical accuracy, efficiency, and reliability.

[0044]

[0025] The automated multifunctional bio-analysis system (100) comprises,

[0045] ■ a single-use disposable cartridge (102) configured for sample processing;

[0046] and

[0047] ■ a portable analyser (103) configured for automated sample movement, incubation, thermal cycling, magnetic manipulation, sonication, optical detection and data analysis.

[0048]

[0026] The cartridge (102) is prefilled with reagents and buffers required for nucleic acid extraction, PCR amplification, TRFIA, and CLIA assays.

[0049]

[0027] The analyser (103) comprises a plurality of integrated functional modules. In an embodiment, the analyser includes a barcode module (70) for cartridge identification and sample tracking, and a cartridge holding mechanism (69) for positioning and securing the cartridge (102). In a further embodiment, the analyser (103) includes a thermal cycler module (76) for temperature-controlled reactions and for precise temperature regulation during PCR and incubation steps; a motor module (55) for actuation of flow-control valves of the cartridge (102), an optical module (62) for detection of fluorescent and chemiluminescent signals, a magnet actuation module (79) for manipulation of magnetic particles, and a sonication module (71) for mechanical disruption of sample material. The analyser (103) further comprises a data processing and analysis module (53) configured to process and interpret assay results. Further, these modules are operatively controlled by a program controller (64) to ensure coordinated and automated operation of the system (100).

[0028] The cartridge (102) comprises three structurally distinct parts, namely an upper part, a lower part, and a well part, which are permanently joined together, preferably by laser welding or an equivalent bonding technique, to form a sealed, single-use fluidic device.

[0050] ■ The upper part comprises a rigid cover layer configured to house and support a sample inlet port (2), an air-pressure activation region including a piercing interface (19), a valve actuation interface (17), and air distribution channels (7, 15). The upper part further comprises mechanical coupling features configured to engage with the cartridge holding mechanism (69) of the analyser (103), thereby enabling precise positioning and controlled valve actuation. The upper part maintains sterility of internal reagents, protects preloaded reagents and prevents evaporation prior to cartridge activation.

[0051] ■ The lower part forms a base layer comprising a microfluidic network of interconnected chambers including: a sample chamber (8), a lysis or reaction chamber (13), one or more reagent chambers (9, 10, 11), and a waste chamber (14). These chambers are fluidically connected by flow channels and are configured for gravity-assisted and / or pressure-assisted fluid movement controlled by valves (3, 4, 5). The lower part further comprises interaction regions configured for engagement with the magnet actuation module (79) and thermal control module (76).

[0052] ■ The well part comprises one or more optically transparent reaction wells (18), including PCR wells and / or detection wells, positioned for thermal contact with the thermal block of the thermal cycler module (76) during PCR cycling, and in optical alignment with the optical module (62) during fluorescence or chemiluminescence detection. The reaction wells (18) are formed from an optical-grade polymer material suitable for signal transmission.

[0053]

[0029] The upper part, lower part, and well part are aligned and permanently bonded to create sealed fluidic pathways, thereby preventing leakage, crosscontamination, and reagent evaporation during operation.

[0030] The said cartridge (102) comprises:

[0054] • a sample chamber (8) containing lysis buffer, lyophilised magnetic beads labelled with antibodies, or dilution buffer such as phosphate-buffered saline (PBS) or normal saline;

[0055] • a chamber (11) containing magnetic beads or an antibody conjugate;

[0056] • reagent chambers (9, 10) containing liquid reagents including wash buffers or assay substrates; and

[0057] • a lysis chamber (13) fluidically connected to the reagent chambers (9, 11, 10, 8) through valve (3); wherein rotation of valve (3) enables selective fluid communication between the interconnected chambers.

[0058]

[0031] The cartridge (102) further comprises flow-control valves (3, 4, 5) configured for controlled routing of fluids between the chambers.

[0059]

[0032] The waste chamber (14) is fluidically connected to the lysis chamber (13) through a valve (4), and is configured to collect liquid waste generated during sample processing, washing, and reagent exchange steps. In a preferred embodiment, the waste chamber (14) contains an absorbent material, such as absorbent paper or porous polymer media, configured to immobilize collected waste liquid and thereby prevent backflow, spillage, and sloshing of waste liquid during cartridge handling, thermal cycling, and disposal.

[0060]

[0033] The cartridge (102) is fabricated from thermostable plastics such polycarbonate (PC), polyethylene (PE), or ABS. The optically transparent portions of the cartridge, including the reactions wells (18), are preferably formed from an optically clear polymer material or polycarbonate.

[0061]

[0034] A vent channel / air distribution channels (15) fluidically connects the waste chamber (14) to a cartridge air cap (20). The air distribution channels (15) serves a dual function:

[0062] (i) allows displacement and release of trapped air during liquid inflow into the waste chamber, thereby preventing pressure buildup that could impede gravity-driven flow; and(ii) enables pressure equalization between the waste chamber (14) and the external environment through the cartridge air cap (20), thereby facilitating smooth transfer of liquid waste while maintaining controlled airflow.

[0063]

[0035] The air distribution channel (15) is dimensioned to permit airflow while substantially restricting liquid flow, thereby preventing leakage of liquid waste into the air cap region and minimizing aerosol formation.

[0064]

[0036] The said cartridge (102) further comprises a plurality of externally accessible grooves formed on its outer surface for interaction with corresponding module of the analyser (103). A positioning groove (6) is configured to engage with the cartridge holding mechanism (69) to ensure correct orientation and repeatable placement of the cartridge (102), thereby enabling accurate alignment of internal chambers with thermal, magnetic, and optical modules. A sonicator engagement groove (28) is configured to enable coupling with a sonicator probe of the sonication module (71) and is positioned adjacent to the lysis chamber (13), thereby enabling transmission of ultrasonic energy into the sample. A magnet positioning groove (22) is configured to align with the magnet actuation module (79) to ensure positioning of a magnet relative to a chamber containing magnetic particles. Further, these grooves are integrally formed as part of the cartridge structure and are positioned such that the corresponding analyser modules engage automatically upon cartridge insertion without requiring manual alignment.

[0065]

[0037] Referring to Figure 1, there is illustrated a schematic representation of the disposable cartridge (102) according to an embodiment of the present invention. The upper structural body (1) forms the top portion of the cartridge (102) and houses the sample inlet port (2), valve actuation interfaces (17) and a Piercing interface (19). The upper body (1) provides mechanical rigidity and protects internal reagent chambers prior to cartridge activation. The positioning groove (6) is formed on the outer surface of the cartridge holding mechanism (69), thereby ensuring proper orientation and alignment relative to the analyser modules. Thesample inlet port (2) is configured to receive a biological sample and is sealable after sample loading to maintain cartridge integrity. The valve actuation interface (17) provides mechanical access for engagement with the motor module (55) to enable controlled rotation of the internal flow-control valves. The vent channel (15) fluidically connects the waste chamber (14) to the cartridge air cap (20) to allow air displacement and pressure equalization during liquid transfer. The piercing interface or air-activation region (19) is configured to cooperate with a piercing mechanism of the analyser to initiate air-assisted fluid movement within the cartridge. A retainer (45) is provided to prevent unintended activation of the air-pressure system during storage and transport.

[0066]

[0038] Referring to Figure 2A, there is illustrated a schematic representation of air-pressure activation and distribution features of the cartridge (102). An air distribution channel (7) distributes air from the cartridge air cap (20) to selected internal chambers upon activation. An area separator (21) is provided within the waste chamber (14) to separate liquid from the air gap region positioned above the separator. Upon insertion of the cartridge (102) into the analyser (103), a controlled actuation mechanism engages the cartridge air cap (20) to activate the air distribution channels (7, 15).

[0067]

[0039] Referring to Figure 2B, there is illustrated a schematic representation of lower and side features of the cartridge (102). The cartridge body (1) encloses internal fluidic chambers and provides structural rigidity. An air channel (27) connects the waste chamber (14) to the air chamber to facilitate pressure equalization. The sonicator engagement groove (28) is positioned adjacent to the lysis chamber (13) for mechanical coupling with the sonication module (71). The magnet positioning groove (22) is positioned adjacent to a chamber (24) containing magnetic particles to align with the magnet actuation module (79). The fluidic channel network (25) interconnects the sample chamber (8), lysis or reaction chamber (13), reagent chambers (9, 10, 11), reaction wells (18) and waste chamber (14). A reagent or reaction chamber region (23) is configured to receiveliquid reagents or function as an incubation chamber. An outlet or air distribution channel (29) directs processed air toward the air chamber through valve (4).

[0068]

[0040] Referring to Figures 3A and 3B, there is illustrated a schematic representation of a channel-splitting architecture provided at a PCR well end of the cartridge (102). Figure 3A depicts an interior view of a fluidic splitting structure and Figure 3B depicts an exterior view of the same region. The structure enables controlled and substantially equal distribution of an eluted nucleic acid solution into multiple PCR wells (18) for multiplex analysis. A primary inlet channel (30) delivers the eluted nucleic acid solution from an upstream lysis or elution chamber (13) toward a PCR distribution region and is configured to provide a substantially uniform flow front prior to splitting. An air outlet channel (29) is provided to direct displaced air toward an air chamber during filling of the PCR wells (18). A channel expansion region or flow equalization chamber (34), positioned downstream of the inlet channel (30), reduces flow velocity and equalizes hydraulic pressure before the fluid is divided into multiple branches, thereby promoting uniform distribution among the PCR wells (18). A primary split junction (28) is formed downstream of the expansion region and divides the incoming liquid stream into multiple secondary flow paths. A plurality of secondary distribution channels (31) extends from the primary split junction, each secondary distribution channel (31) being dimensioned to provide substantially identical hydraulic resistance to promote substantially equal volumetric flow into respective downstream PCR wells (18). PCR well inlet ports (32) provide fluidic communication between respective secondary distribution channels (31) and corresponding PCR wells (18), the inlet ports (32) being configured to direct liquid into the PCR wells substantially without air entrapment. An air vent structure or capillary stop feature (33) is associated with each PCR well inlet (32) to allow displaced air to escape during filling while substantially preventing backflow and overfilling, thereby promoting reproducible filling of each PCR well prior to thermal cycling.

[0041] Referring to figure. 4, there is illustrated a retainer plastic component (36) configured to secure the piercing spring unit (41) in position within the cartridge (102). A retainer plastic is configured to be pressed onto the cartridge body before loading the cartridge (102) with sample (101), thereby locking the piercing housing (37) and piercing spring unit (41) in a pre-activation state and establishing an air-sealed configuration. The attachment junction (35) represents the connection interface between the cap and the retainer component (36), thereby securing the assembly during storage and transport.

[0069]

[0042] Referring to Figures 5A and 5B, there is illustrated a top cover (45a) of the cartridge (102) configured to prevent piercing when the cartridge is not activated, and an associated air chamber assembly (41). The top cover (45a) comprises a plurality of hollow piercing pins (40) configured to pierce a sealing membrane of a cartridge chamber (37), and a spring mechanism (38) adapted to maintain the piercing pins (40) in an unpierced state until activation. A sample inlet opening (2) is fluidically connected to the sample chamber (8) through a connection port (39). Figure 5B further illustrates the air chamber assembly (41) which is configured to provide positive pressure to one or more reagent chambers. The air chamber assembly (41) comprises the ports (40) configured for air entry. The top cover piercing cap assembly (45) further comprises one or more locking features or flip-lock elements (38) positioned on opposing sides of a piercing housing (37). The locking features (38) are configured to restrain the piercing assembly in a preactivation position and prevent unintended movement of the piercing pins (40), thereby maintaining reagent integrity and preventing premature air ingress or evaporation prior to cartridge use. Upon insertion of the cartridge (102) into the analyser (103), an actuation force is applied to the piercing housing (37), overcoming a spring force of the spring mechanism (39) and causing the hollow piercing pins (40) to pierce the sealing membrane, thereby establishing fluidic communication between the air chamber (41) and air distribution channels (7, 15). The air chamber (41) functions as a pressure reservoir allowing ambient air to enter and distribute through the air distribution channels (7, 15) to internalchambers including reagent chambers and the waste chamber (14), thereby enabling pressure equalization and assisting gravity-driven liquid flow through a microfluidic network within the cartridge (102). In the activated configuration, the locking features (38) retain the piercing assembly in a pierced position, preventing retraction of the piercing pins (40) and maintaining a stable air pathway throughout assay execution and promoting smooth and complete liquid transfer, preventing vacuum formation, minimizing bubble entrapment, and supporting reliable cartridge operation without the use of external pumping mechanism.

[0070]

[0043] Referring to figure. 6 there is illustrated a schematic representation of the transparent PCR well (18). The said transparent well (18) comprises a plurality of slots (44), for optical reading. The well (18) is joined to said cartridge body (102) by laser welding. In an embodiment, said transparent well (18) comprises four number of slots (44) for optical reading. Each slot (44) can be pre-filled with primer, probes, and master mix in case of PCR cartridge, or with substrate solution in case of CLIA cartridge. In another embodiment, the well (18) of the PCR chamber may contain primers, probes, enzymes, detergents, blocking agents, dyes, markers, or other reagents suitable for detecting a target or reference nucleic acid sequence. The PCR wells (18) are maintained under vacuum to ensure equal distribution of the elute from the lysis chamber, with each well receiving approximately 50 pL of elute. In one embodiment the PCR well (18) will have lyophilised master mix containing optimised quantities of primers and probes, wherein each well comprises a specific primer and probe for an internal control, typically beta-actin. In another embodiment, one well is used as a negative control, containing no template primers and probes, while another well is prefilled with plasmid having the gene of interest along with primer and probes to function as a positive control. The vertical part (43) covers the channels in the cartridge body enabling flow of reagent to the wells (44).

[0071]

[0044] Referring to figure. 7 there is illustrated a schematic representation of the retaining block (45) fixed on the cartridge top. The retaining block (45) preventsthe piercing cap from breaking the sealing barrier during storage and transportation. During activation stage, the retaining block (45) is pulled out from the cartridge top, allowing the piercing cap to press and pierce the sealing membrane comprising aluminium seal to activate the air channels.

[0072]

[0045] Referring to figure. 8 illustrates a schematic representation of the flow control valve (3, 5). The said flow control valve (3, 5) are direct valves with a through hole for allowing liquid flow from the sample chamber (7). The rotation of the valve (3, 5) is controlled by the motor module (55). Each valve body (46) comprises a through-hole (47) with 2mm diameter for connecting chambers and a slit (48) for engaging the rotating actuator.

[0073]

[0046] Referring to Figure 9, there is illustrated a schematic representation of the angular flow control valve (4) according to an embodiment of the present invention. The valve (4) comprises a valve body (49) having a rotatable valve core (50) provided with a primary through-hole and an auxiliary vent hole (51). The primary through-hole selectively aligns with a liquid transfer channel connecting the lysis chamber (13) to the PCR well (18) to permit controlled transfer of liquid into the PCR well when the valve (4) is in an open position. The auxiliary vent hole (51) establishes temporary fluidic and pneumatic communication between the liquid transfer channel and a vent channel communicating with the waste chamber (14) and further with the air chamber assembly (42), thereby enabling displacement of trapped air during liquid transfer and facilitating smooth and complete filling of the PCR well (18) without bubble formation. Upon completion of liquid transfer, the motor module (55) rotates the valve core (50) to a closed position, wherein both the primary through-hole and the auxiliary vent hole (51) are misaligned from their respective channels, thereby sealing the liquid transfer channel and the vent channel to prevent backflow, air ingress, and evaporation from the PCR well (18) during subsequent thermal cycling and detection steps.

[0047] Referring to Figure 10, there is illustrated a schematic representation of the automated multifunctional bio-analyser (103) according to an embodiment of the present invention. The analyser (103) comprises an external casing (52) enclosing functional modules including, barcode module (70), cartridge holding mechanism (69), thermal control module (93), motor module (55), fluorescence detection module (91), and sonicator module (71). These modules are controlled by the program controller (64) and linked to a data processing and analysis module (53). In a preferred embodiment, said cartridge (102) is inserted into the cartridge insertion slot (54) via a linear stepper motor. The motor module (55) actuates flow control valves (3, 4, 5) with shafts and gears aligned to engage valve features accurately. Further, the home position of motors is preprogrammed to allow accurate alignment and rotation angles for valve operation.

[0074]

[0048] Referring to Figure 11, there is illustrated an exploded view of a cartridge holding mechanism (69) according to an embodiment of the present invention. The cartridge holding mechanism (69) is configured to receive, position, secure, and align the disposable cartridge (102) within an analyser (103) such that cartridge chambers are accurately positioned relative to the thermal control module, magnetic actuation module, valve actuation module, and optical detection module. The cartridge holding mechanism (69) comprises a base support frame (65) fixed within the analyser chassis to provide structural rigidity to the cartridge holding assembly (69). A cartridge guide rail or alignment frame (66) is mounted on the base support frame (65) to guide the cartridge (102) along a predefined insertion path, thereby ensuring repeatable and accurate positioning during insertion. A cartridge locking bracket (67) is provided to mechanically secure the cartridge (102) in an operational position after insertion to prevent movement during valve actuation, thermal cycling, and optical detection. The cartridge holding mechanism (69) further interfaces with the program controller (64) configured to coordinate actuation of associated modules once the cartridge (102) is locked in position. A spring-loaded pressing plate (73) is arranged above or adjacent to the cartridge (102) and is configured to apply a controlled downwardforce on the cartridge body to ensure intimate thermal contact between cartridge chambers and the thermal control module, as well as stable alignment with optical and magnetic modules. The cartridge holding mechanism (69) further comprises one or more actuation pins or engagement posts (96) configured to engage corresponding grooves or features formed on the cartridge (102) to ensure correct orientation and prevent incorrect insertion, and one or more end-stop elements or positioning stops (97) configured to define a final seated position of the cartridge (102) within the cartridge holding mechanism (69), thereby ensuring consistent spatial alignment across multiple cartridges.

[0075]

[0049] Referring to figure. 12, illustrates a schematic representation of the analyser (103) along with plurality of modules including, cartridge loading mechanism (68), cartridge carrier (69), barcode module (70), thermal cycler module (76), magnet actuation module (79), valve actuation module (72), sonication module (71), optical module (75), data processing module (78), display unit (80), optical fibers (72) and linear stage assembly (74) for positioning the optical module (75). The said thermal cycler module (76) is located at the base portion of said analyser (103) and comprises a heat sink coupled with a Peltier device and an aluminium block configured to conduct thermal cycling. A temperature sensor is provided to transmit temperature data from the aluminium block to the control module for regulating temperature cycling. In an exemplary embodiment according to the present invention, said temperature sensor is selected from PT 1000 sensor for precise control of Peltier-based temperature cycling. The said magnet actuation module (79) comprises a linear actuator (79A) connected to a neodymium magnet, for capturing and releasing magnetic beads within the lysis chamber (13). The said neodymium magnet is attached to the shaft of said linear actuator (79A), which is mounted on two slides using a linear slide on either side.

[0076]

[0050] Referring to Figure 12A, there is illustrated a sonicator and magnet actuator assembly (120) forming an integrated part of the analyser (103) andcorresponding to the sonication module (71) and magnet actuation module (79). The analyser (103) comprises the sonication module (71) and the magnet actuation module (79) arranged adjacent to the cartridge holding mechanism (69). The assembly comprises a NEMA 14 stepper motor (70a), a lead screw (70b), a linear rail (70c), and a linear slide (70d), which together form a linear actuator mechanism corresponding to the actuator (79 A) driving the magnet actuation module (79). The sonicator holder (70e) and a sonicator probe (70f) correspond to the sonication module (71) and are positioned to interface with the cartridge at the sonicator engagement groove (28). The magnet holder (79a) carrying a neodymium magnet (79b) corresponds to the magnet actuation module (79) and is positioned to align with the magnet positioning groove (22) of the cartridge. In this configuration, Figure 12A illustrates functional placement and spatial coordination of the modules within the analyser, while the detailed assembly drawing illustrates the mechanical construction enabling such operation. The magnet actuation module (79) is positioned adjacent to cartridge chambers and thermally isolated from heater plates (84, 85, 86). Linear translation of the magnet holder (79a) via the actuator mechanism advances the neodymium magnet (79b) toward a selected cartridge chamber to immobilize magnetic particles contained therein, and retraction of the magnet reduces the magnetic field at the chamber to permit resuspension of the particles in a liquid medium, thereby enabling automated execution of magnetic bead-based binding, washing, and elution steps. Retraction and advancement of the magnet relative to the cartridge chambers enable controlled immobilization and release of magnetic particles without interfering with thermal zones. The sonicator probe (70f) is positioned to interface with a cartridge wall adjacent to the lysis chamber while remaining spatially separated from heater plates, consistent with the thermal isolation illustrated in Figure 12A, thereby demonstrating coordinated integration of magnetic actuation, sonication, and thermal control within the analyser.

[0077]

[0051] Referring to Figure 13, there is illustrated a schematic exploded view showing the interaction between the magnet actuation module (79), the thermalinterface region, and the cartridge positioning zone within the analyser (103), demonstrating the spatial coordination of magnetic manipulation and temperature control while maintaining thermal isolation and mechanical stability. The cartridge (102) is configured to slide into the analyser (103) through the cartridge holding mechanism (69) such that specific cartridge chambers align with a plurality of heater plates (84, 85, 86) mounted on the analyser body plate. Each heater plate is configured to independently regulate the temperature of a corresponding cartridge chamber, thereby enabling different incubation or reaction temperatures within the same cartridge. The heater plates (84, 85, 86) being mounted on a support plate (83) to provide structural stability and ensures uniform thermal contact, and being thermally coupled to temperature sensors for real-time feedback to the thermal control module for precise temperature regulation. The region between adjacent heater plates (84, 85, 86) is thermally isolated using an insulating plate (82), preferably formed of an epoxy-based or polymeric insulating material, to prevent lateral heat transfer between adjacent heater zones, thereby avoiding thermal cross-talk and ensuring accurate temperature control of individual cartridge chambers. The coordinated arrangement of heater plates, insulating structures, and the magnet actuation module allows simultaneous execution of temperature-controlled reactions and magnetic particle manipulation within the cartridge (102), thereby enabling fully automated assay workflows for PCR, TRFIA, and CLIA. The NEMA stepper motor (81, 83) are used for actuation of the cartridge valves and are engaged using a shaft (82).

[0078]

[0052] Referring to figure. 14 illustrates a schematic representation of the optical assembly (62) according to an embodiment of the present invention. The said optical assembly (62) is configured to emit light at required wavelengths and to collect emitted or generated light from the reaction well (18). The said optical assembly (62) comprises, a plurality of optical fibers (95) connected to a fiber holder (87) to facilitate transmission of light to and from the optic assembly (88), along with a detector module (90) for chemiluminescent light detection. In anembodiment, the optical module (62) is equipped with a UV LED module (89) to emit light at approximately 350nm for fluorescence immunoassay (FIA). In another embodiment, the optical module (75) includes a dichroic assembly (88a) and a plurality of monochromatic LEDs (88b) providing excitation wavelengths in the ranges of approximately 460-480 nm, 520-540 nm, 570-590 nm and 620-645 nm. The selected LEDs have power ratings in the range of 1-3 W and compact dimensions of approximately 3.5 mm x 3.5 mm with a height of about 2 mm. A lens assembly comprising a first lens (LI) and a second lens (L2) is arranged such that the first lens (LI) collects and collimates light emitted from the LED. The viewing angle of the LEDs, defined as the angle at which the intensity becomes half of its peak intensity, is approximately 120°, and therefore a short focal-length lens is used to effectively collect and collimate the emitted light. The collimated light passes through a bandpass filter (88c) positioned between the two lenses and is then focused by the second lens (L2) onto the optical fiber (95). The lenses (LI, L2) are oriented such that the flat surfaces face the LED and the optical fiber respectively. Further, since both the lenses are identical, the magnification is approximately 1, although it may be varied by slightly adjusting the relative position of the lens assembly. The configuration ensures that light passes substantially perpendicular to the bandpass filter to maintain spectral accuracy. The optic module (75) includes four emission filters within the module. The optical module (75) includes excitation filters of 465 + 15nm, 527 + 8nm, 571 + 9nm and 637 + 12nm, and corresponding emission wavelengths of 510 + lOnm, 564 + 9nm, 612 + 9nm and 685 + 15nm, representing the following dyes for labelling the probes: FAM, HEX / VIC, ROX and Cy5 dyes respectively. A photodiode (88d) is provided for light detection. In an embodiment, the optical fibers (95) have a core diameter of approximately 0.6 mm with 0.06mm cladding and a numerical aperture (NA) of 0.22, corresponding to a half divergence angle of approximately 13°. The optic assembly is mounted on a linear stage (91) configured to enable the optic assembly to move across the four optical fibers.

[0053] Referring to Figure 15, there is illustrated a schematic representation of the thermal block assembly (59) configured for polymerase chain reaction (PCR) within the automated multifunctional bio-analyser (103). The thermal block assembly (59) is designed to provide precise, rapid, and uniform temperature control to the PCR reaction wells (18) of the cartridge (102) during thermal cycling. The thermal block assembly (59) comprises a thermally conductive block (93), preferably fabricated from aluminum or an aluminum alloy, having a plurality of recesses or contact surfaces configured to correspond with the geometry and position of the transparent PCR wells (18) formed in the cartridge (102). The conductive block (93) ensures uniform heat transfer across all PCR wells, thereby minimizing temperature gradients between reaction chambers. The thermally conductive block (93) is operatively coupled to a thermoelectric element (94), preferably a Peltier device, configured to selectively generate heating or cooling upon application of electrical current. By reversing the polarity of the applied current, the Peltier element (94) enables rapid switching between heating and cooling modes, thereby supporting fast PCR ramp rates. The Peltier element (94) is thermally coupled to a heat dissipation assembly (95) comprising a heat sink and, optionally, an active cooling fan. The heat dissipation assembly (95) is configured to efficiently remove excess heat generated during thermal cycling and to maintain temperature stability and improve cycling accuracy. In operation, when a PCR-configured cartridge (102) is positioned within the analyser (103), the cartridge holding mechanism (69) ensures intimate thermal contact between the PCR wells (18) and the thermally conductive block (93). The program controller (64) drives the Peltier element (94) according to predefined thermal cycling parameters, including denaturation, annealing, and extension temperatures and corresponding dwell times. Temperature feedback is provided by one or more temperature sensors (96), preferably selected from resistance temperature detectors (RTDs) such as PT 1000 sensors, embedded within or positioned adjacent to the thermally conductive block (93). The feedback signals are processed by the thermal control module to dynamically adjust the power supplied to the Peltier element (94), thereby maintaining precise temperaturecontrol throughout the PCR process. The thermal block assembly (59) is mechanically isolated from adjacent modules and thermally insulated where required to prevent heat transfer to non-PCR regions of the cartridge (102). This configuration ensures stable and repeatable PCR performance while allowing simultaneous execution of non-PCR processes within other regions of the cartridge. The thermal block assembly (59) thereby enables accurate and reproducible amplification of nucleic acids, supporting real-time fluorescencebased detection and multiplex PCR assays within the automated multifunctional bio-analyser system.

[0079]

[0054] Referring to figure. 16 illustrates a functional block diagram representing the workflow of the automated multifunctional bio-analyser (100). The system (100) is equipped with several process modules, including a Barcode Module, Thermal Control Module, Motor Module, Fluorescence Module, and Sonicator Module, all operatively controlled by a Program Controller and linked to a Data Processing and Analysis Module to ensure precise and automated operation. In an embodiment, the embedded electronics system and software architecture are implemented in accordance with the block diagram figure 16, wherein an embedded microcontroller communicates with the optical module, thermal cycler module, and mechanical module based on predefined parameters. The platform further comprises a built-in computer for data analysis and storage.

[0080]

[0055] In operation, a specimen is introduced into the system (100) via the sample inlet port and the cartridge (102) is inserted into the analyser. The sample parameters are determined either by scanning the barcode, using the barcode module via UART communication or through user input via the user interface (UI) connected through RS232 or USB. Upon identification of sample parameters, the sample undergoes a meticulous cell lysis and nucleic acid extraction using both chemical and mechanical methods, wherein an enhanced lysis buffer facilitates chemical disruption and mechanical disruption is achieved through sonication and bead beating with zirconium beads. The prefilled reagents mixed with the sample are heated to a predefined temperature for specific duration usinga preheated block. The sample lysis and nucleic acid extraction are done by valve rotation and magnetic bead insertion, with the beads excited by the sonicator probe, which is controlled by the sonication module through GPIO serial communication. The valve rotation and piston movement are controlled by the motor module, which also communicates via GPIO. The resultant obtained after nucleic acid extraction is transferred to a first PCR chamber (PCR1) for thermal cycling comprising denaturation, annealing, and extension for a predefined number of cycles and duration, carried out by the thermal cycler module through GPIO serial communication. The resultant from the PCR1 is subsequently mixed with a resuspension buffer and is transferred to a second PCR chamber (PCR2), where it undergoes additional thermal cycling and fluorescence detection, controlled by the fluorescence module through I2C communication. The fluorescence intensity of the sample, obtained as per the imposed light is recorded and analysed by the fluorescence module. The data obtained by the fluorescence module is further analysed by the data analysis module and the resultant graph is plotted on the UI interface. This entire process is controlled and monitored by the Program controller and the results are displayed on the UI. In an embodiment, the automated multifunctional bio-analyser (103) further comprises a software analysis module executed by the program controller (80), the software analysis module being configured to process raw optical signals generated during PCR, CLIA, and FIA assays and to convert said signals into validated diagnostic outputs. The software analysis module receives time-resolved or intensity-based signal data from the optical detection module and performs signal preprocessing, including background subtraction, baseline normalization, temporal alignment, and noise filtering. For fluorescence-based assays, spectral cross-talk compensation and channel-specific normalization may be applied to ensure accurate discrimination between multiple fluorophores. In PCR embodiments, the software analyses cycle-by-cycle fluorescence data to generate amplification curves for each parameter. A cycle threshold (Cq) or equivalent metric is calculated using predefined curve-fitting or thresholding methods, and amplification efficiency, curve shape, and signal growth kinetics are evaluated toconfirm reaction integrity prior to result reporting. In CLIA and FIA embodiments, the software analyses end-point or time-resolved signal intensities obtained from the detection wells. Signal values are optionally corrected using reference measurements or calibration factors stored in the system memory. Quantitative concentrations or qualitative results are determined by comparison with stored calibration curves, lookup tables, or predefined decision thresholds. Each analysed signal is correlated with its corresponding spatial location (reaction well) and optical identifier (fluorophore, luminescent label, or time-resolved window) to uniquely associate the signal with a specific target or control parameter. Following signal analysis, the software integrates the analytical results with the control validation logic, wherein outputs are reported only when internal control, positive control, and negative control acceptance criteria are satisfied. The software further assigns confidence metrics, flags atypical signal patterns, and stores raw and processed data for traceability and audit purposes, and the final diagnostic output is displayed as numerical values, qualitative interpretations, graphical plots, or combinations thereof, and may be stored locally or transmitted to an external information system. This software-based analysis ensures accurate, reproducible, and fully automated interpretation of assay results across PCR, CLIA, and FIA workflows.

[0081]

[0056] Referring to figure. 17, the optic fiber arrangement for the imaging system is illustrated. In an embodiment a detector module (90) is used for chemiluminescent light detection. In a further embodiment, the emitted fluorescence from each well is detected using a CMOS camera. Each well contains 600-micron optical fibres, and for 4 wells, a total of 4 fibres are arranged such that emitted light passes through corresponding emission filters, as shown in figure 17. A highly sensitive monochrome camera having a sensor size of approximately 26mm x 15mm may be used to image all fibres simultaneously. In one embodiment, a CMOS camera incorporating a SONY IMX571BLR-J sensor, having an effective area of 23.4 mm x 15.6 mm, is configured to capture images of the entire fiber bundle on a single sensor. Alternatively, a camera incorporatinga SONY IMX183CQJ-J sensor with a sensor size of 13.1mm x 8.8mm (for example, e ASH 83) may be used, wherein two imaging modules are employed to capture the entire array. In another embodiment, two MX042MR-GP-BSI-X4G2-FV modules from Ximea, each being a 4.2MP uncooled CMOS camera, are used for imaging the fiber output. In a further embodiment, four separate detectors are provided, one for each well, each detector being optically coupled to 24 fibre ends.

[0082]

[0057] Referring to figure. 18, the arrangement of the optical fibers for the photodetector unit is illustrated. In an embodiment, detection is performed using a highly sensitive silicone photodetector, SI 6008-66 from Hamamatsu as shown in figure 18. The photodetector comprises a photosensitive area of approximately 5.8 mm x 5.8 mm, a maximum dark current of 50pA, and a spectral response range of 380nm to 1100 nm. A dome assembly is provided, comprising five fibre ends together with a 4mm x 4mm emission filter and a focussing lens. The five fibres from each well are arranged within the dome assembly such that emitted light is focussed onto the 5.8 mm x 5.8 mm photosensitive area of the detector.

[0083]

[0058] In the same preferred embodiment according to the present invention, said cartridge (102) is a self-contained, air pressure-assisted gravity-flow microfluidic cartridge configured to perform sample processing, nucleic acid extraction, thermal cycling, incubation, chemical reactions, washing steps and optical detection within a sealed environment.

[0084]

[0059] In one embodiment, the PCR cartridge comprises a plurality of reaction wells (18), wherein each reaction well (18) is preloaded with assay-specific primers and probes corresponding to one or more target nucleic acid sequences. Each reaction well (18) may further include primers and probes for internal controls, positive controls, or reference targets.

[0060] In another embodiment, multiplexing is achieved by incorporating multiple fluorescently labelled probes within one or more PCR wells (18), wherein each probe is tagged with a fluorophore having a distinct excitation and emission wavelength. The multiplex optical system (62) is configured to excite and detect fluorescence signals from a plurality of spectrally distinguishable fluorophores, thereby enabling simultaneous detection of multiple PCR targets from the same reaction volume.

[0085]

[0061] In the same preferred embodiment, up to sixteen parameters are detected comprising:

[0086] (i) target-specific nucleic acid sequences corresponding to different pathogens, genes, or mutations;

[0087] (ii) one or more internal amplification controls to verify PCR efficiency; (iii)one or more positive controls; and

[0088] (iv)one or more negative or reference controls.

[0089]

[0062] The analyser (103) sequentially or concurrently acquires fluorescence data from the plurality of PCR wells (18) using the multiplex optical module (62) and processes the data to independently quantify each parameter based on its spatial location and / or spectral signature. This configuration enables high-throughput, multi-target nucleic acid detection within a single automated PCR run without increasing assay time or sample volume.

[0090]

[0063] In immunoassay embodiments, including TRFIA, CLIA and FIA, the detectable parameters correspond to multiple analytes and controls distributed across multiple detection wells and / or multiple optical labels, thereby enabling simultaneous multi-analyte detection and assay validation.

[0091]

[0064] The multiplex optical system (62) is configured to independently process signals corresponding to the plurality of parameters, thereby enabling accurate multiplex diagnostics within a single automated run.

[0065] In one embodiment according to the present invention, said cartridge (102) configured for PCR analysis comprises:

[0092] a sample chamber (7) containing a lysis buffer composition, having 1-10% NaOH, guanidine isothiocyanate (GITC) and Tris HIC, or lyophilized magnetic beads labeled with antibodies, to enable efficient cell lysis for PCR analysis;

[0093] a magnetic bead chamber (11) containing magnetic beads suspended in ethyl alcohol for nucleic acid extraction;

[0094] a plurality of reagent chambers (9, 10, 8) containing wash buffers, elution buffers or other substrates / reagents for use in various procedures associated with the PCR analysis; and

[0095] a lysis chamber (13) fluidically connected to said sample chamber (7) through a valve (3), allowing controlled liquid flow from the sample chamber (7) to the lysis chamber (13) upon rotation of the valve, to facilitate the lysis process.

[0096]

[0066] In an exemplary embodiment according to the present invention, said cartridge (102) configured for PCR analysis, said sample chamber (7) contains approximately 1% NaOH and 3% GITC; the lysis chamber (13) contains lyophilised ball of Proteinase K and Poly A to enhance lysis efficiency and RNA recovery; chamber (11) contains magnetic beads of approximately lOOnm diameter in ethyl alcohol; and chamber (9) contains a wash solution comprising Tris-HCL or PBS with Tween 20.

[0097]

[0067] In some embodiments, said chamber (9) contains nuclease-free water or PBS-T as a wash buffer.

[0098]

[0068] In certain embodiments, said chamber (10) contains nuclease-free water for elution of DNA. In CLIA configurations, chamber (10) may alternatively contain a stopping reagent.

[0069] In another embodiment according to the present invention, a cartridge (102) for analysing a sample (101) using time-resolved fluorescent immunoassay (TRFIA) comprises,

[0099] a sample chamber (7) containing lyophilized antibody-coated magnetic beads;

[0100] chamber (9) containing a wash buffer with PBS-T;

[0101] chamber (11) containing fluorescent beads labelled with antibodies; chamber (10) containing a wash buffer; and

[0102] chamber (8) containing a dilution buffer with PBS.

[0103]

[0070] In yet another embodiment according to the present invention, a cartridge (102) for analysing a sample (101) using a chemiluminescent immunoassay (CLIA) comprises,

[0104] a sample chamber (7) containing magnetic particles labelled with a primary antibody;

[0105] chamber (9) containing a wash buffer;

[0106] chamber (11) containing secondary antibody HRP conjugate, chamber (8) containing a wash buffer;

[0107] chamber (10) containing a substrate solution; and

[0108] chamber (18) containing a stop solution.

[0109]

[0071] In the same preferred embodiment according to the present invention, said flow control valve (3, 5) are direct through-hole rotary valves, and valve (4) is an angular valve.

[0110]

[0072] In the same preferred embodiment according to the present invention, said cartridge (102) is sealed with an aluminium foil using heat sealing after reagent filling to prevent leakage and contamination.

[0073] In the same preferred embodiment according to the present invention, a spring holder (34) is positioned over the aluminium seal as a press-fitting module to secure a piercing air chamber (37).

[0111]

[0074] In the same preferred embodiment according to the present invention, air channels extend from chambers (12, 13, 14) to chamber (20). Upon piercing of the chamber seal by part (40), an air connection is established to facilitate gravity-assisted flow. Further, a flip-lock mechanism (38) on both sides, prevents reverse movement of the piercing assembly.

[0112]

[0075] In the same preferred embodiment according to the present invention, said barcode module (70) comprises, a barcode reader configured to read QR code data printed on the cartridge (102), and share the information with the analyser (103). The QR code may include data related to the cartridge identification name, assay protocol and manufacturing information.

[0113]

[0076] In an exemplary embodiment according to the present invention, the valves (3, 4, 5) are actuated using NEMA 17 stepper motors with encoders.

[0114]

[0077] In an exemplary embodiment according to the present invention, the sonication frequency is fixed at 25kHz, applied in short-duration pulses of 10 sec with 10 sec intervals.

[0115]

[0078] In the same preferred embodiment according to the present invention during PCR processing, the sample (101) is introduced into sample chamber (7) containing a chemical lysis solution. The mixture is then transferred to the lysis chamber (13) through valves (3, 5) by timely rotation. The lysis chamber (13) contains lyophilised Proteinase K, Poly A and Zirconium beads, and sonication is applied to enhance lysis. Once lysis is complete, magnetic beads are mobilised into the chamber and allowed to react for a specific time. The magnet is actuated to hold the beads with bound nucleic acids, while the bead-free solution isreleased to the waste chamber (14) through valve (4). Subsequently, wash buffer 1 and wash buffer 2 are sequentially introduced into the lysis chamber (13) and then discharged to the waste chamber to ensure thorough washing. After washing, the elution buffer is mobilised to the lysis chamber (13), and the eluted nucleic acids are collected in the PCR wells (18).

[0116]

[0079] In some embodiments, the extraction of nucleic acid is facilitated by a combined approach: enhanced lysis buffer efficiently breaks down cellular structures to release nucleic acids; followed by bead beating, which further disrupts cell walls and tissues for thorough lysis; and sonication aids in homogenization and fragmentation of the sample (101), enhancing the release of nucleic acids. After these initial steps, magnetic bead-based extraction selectively captures and purifies nucleic acids from the lysate. Magnetic beads are coated with nucleic acid-binding agents, allowing efficient separation of nucleic acids from contaminants and debris.

[0117]

[0080] In some embodiments, when using probe-based qPCR, satisfactory results are often obtained using final concentrations of both primers at 500 nM and the probe at 250 nM, paricularly if the PCR target is abundant and maximum sensitivity is not required. A final concentration of 250 nM probe is commonly used for most assays, but optimization is key to ensure both cost-efficiency and performance. To achieve optimal probe conditions, the probe can be tested at several concentrations, typically ranging from 50 to 500 nM, in combination with the optimized primers.

[0118]

[0081] In one embodiment, the annealing temperature of a qPCR assay is optimised to ensure reaction specificity, especially for multiplex assays. Setting the annealing temperature too low may result in nonspecific amplification, while setting the annealing temperature too high may reduce target yield. Even after calculating the primer Tm, of a primer, it may be necessary to empirically determine the annealing temperature by testing a range of temperatures above andbelow the calculated Tm. Similar testing may be required to optimize the denaturation temperature. The optimal annealing temperature is identified as that which produces the lowest Cq without nonspecific amplification.

[0119]

[0082] In an exemplary embodiment according to the present invention, the PCR master mix used in the cartridge (102) is selected from PCR Buffer IX, dNTP Mix 200 pM each, MgCh 1.5 mM, DNA Polymerase 0.5 -1.0 U / pL, primers and probes and a lyostabilizer up to 50 pL.

[0120]

[0083] In one embodiment, it takes approximately 200 ms to detect each sample tube, during which five raw fluorescence data points are collected and averaged to obtain the final fluorescence value. The fluorescence values of each cycle are plotted as real-time PCR curves for further analysis. Since the system may operate on a different scale compared to a reference system, normalization of the acquired data is necessary. In qPCR, the fluorescence signal from the first few cycles is typically used to determine the baseline fluorescence, serving as the background signal. To address differences in background across equipment, baseline correction is a crucial step in qPCR analysis and is generally integrated into the system. Accordingly, the fluorescence detected in the proposed system is corrected according to baseline determined from the initial five cycles of PCR and scaled to match the RFU of the 40th cycle from the reference system. The resulting plot, shown in figure 19, demonstrates that the proposed system delivers comparable results to the reference system.

[0121]

[0084] In a preferred embodiment according to the present invention, the cycle threshold (Cq) for both the proposed and reference system is obtained by comparing the logarithm of the acquired fluorescence values to a predefined threshold. In other words, the Cq is defined as the cycle number at which the logarithmic fluorescence curve intersects the predefined threshold.

[0122]

[0085] In a preferred embodiment according to the present invention, the relative fluorescence units (RFU) are estimated using the formula,RFU= [(Fluorescence signal at cycle n - Baseline fluorescence) / (Baseline fluorescence)] *100

[0123] where, n represents the cycle number for which the RFU is calculated.

[0124]

[0086] In the same preferred embodiment according to the present invention, the baseline fluorescence is estimated by measuring the fluorescence intensity value from cycles 1-5, with and without the sample. The threshold is set using algorithms that identify the point at which the signal deviates from background noise.

[0125]

[0087] In some embodiments, the CT value which represents the cycle number at which the fluorescence signal crosses a predefined threshold, is automatically calculated by the data processing module. This value corresponds to the point at which the amount of amplified target nucleic acid becomes detectable above background levels. The fluorescence signal is measured at the end of each amplification cycle, and the CT value is determined by interpolating between the two signal measurements between which the threshold was crossed. The cut-off for the CT value is fixed based on analysis of negative and positive samples to ensure accurate detection.

[0126]

[0088] In one embodiment, each reaction well (18) is excited using a ultraviolet light at a wavelength of 360nm, and the emitted light at 610 nm is detected by the detector for performing time-resolved fluorescence analysis using lanthanide-labelled particles.

[0127]

[0089] In an embodiment, the sample chamber (7) of the cartridge contains magnetic beads coated with antibodies. The lysis chamber (13) contains dried lanthanide-labelled beads coated with secondary antibodies. The antigen in sample binds with the magnetic bead antibodies and are transferred to the lysis chamber (), where it is captured by the lanthanide-labelled secondary antibodies to form an immunocomplex. The mixture is incubated and subsequently washed with a wash buffer while the magnetic particles are immobilized using a magnet.The washed beads are then resuspended in a stable buffer, such as PBS, and transferred to the detection well for signal acquisition.

[0128]

[0090] In another embodiment, sample chamber (7) contains a chemiluminescent substrate, such as luminol. Magnetic beads coated with primary antibodies are mixed with the sample, followed by addition of HRP-labelled secondary antibodies. After washing, the magnetic beads are transferred to wells containing the luminol substrate. The enzymatic reaction produces chemiluminescence, which is detected directly by photodiode detectors without the need for optical filters.

[0129]

[0091] EMBODIMENT A:

[0130] Step-by-step process for PCR-Based Nucleic Acid Detection

[0131] In one embodiment, the automated multifunctional bio-analyser system (100) is configured to perform nucleic acid extraction and real-time polymerase chain reaction (PCR) amplification within a single disposable cartridge processed by the analyser.

[0132] • Step Al: Cartridge Identification and Initialization: An assay-specific PCR cartridge (102) is inserted into the analyser (103). The barcode module identifies the cartridge type and associated assay protocol. Upon identification, the analyser automatically initiates a predefined PCR workflow.

[0133] • Step A2: Sample Loading: A biological sample containing nucleic acids is introduced into the sample chamber (7). In one embodiment, the sample chamber contains a chemical lysis solution configured to lyse cells or viral particles and release nucleic acids.

[0134] • Step A3: Chemical and Mechanical Lysis: The sample is transferred to the lysis chamber (13) by valve actuation. Chemical lysis is performed using the lysis solution. Mechanical lysis may be enhanced by: sonication using the sonication module, and / or bead-assisted agitation using mechanical disruption beads present in the lysis chamber.• Step A4: Magnetic Bead-Based Nucleic Acid Binding: Magnetic beads are introduced into the lysis chamber and incubated to allow released nucleic acids to bind to the bead surface.

[0135] • Step A5: Magnetic Immobilization and Waste Removal: The magnet actuation module immobilizes the magnetic beads. The bead-free lysate is directed to the waste chamber (14).

[0136] • Step A6: Washing: One or more wash buffers are sequentially introduced into the lysis chamber from wash buffer chambers (9, 10). After each wash cycle, magnetic beads are immobilized and the wash liquid is transferred to the waste chamber.

[0137] • Step A7: Elution: An elution buffer is introduced to release nucleic acids from the magnetic beads. The eluted nucleic acid solution is transferred into PCR reaction wells (18).

[0138] • Step A8: PCR Thermal Cycling: The thermal block assembly subjects the PCR wells to controlled thermal cycling comprising denaturation, annealing, and extension steps. Each PCR reaction well contain lyophilized master mix, primers, probes, and internal controls.

[0139] • Step A9: Real-Time Fluorescence Detection: During amplification, fluorescence signals are excited and detected using the multiplex optical system. Fluorescence data is acquired for each cycle and processed to generate amplification curves and cycle threshold (Cq) values.

[0140]

[0092] EMBODIMENT B:

[0141] Step-by-step process for Time-Resolved Fluorescence Immunoassay (TRFIA / FIA)

[0142] In another embodiment, the system is configured to perform time-resolved fluorescence immunoassay.

[0143] • Step Bl: Cartridge Identification: A TRFIA-configured cartridge is inserted and identified by the analyser.• Step B2: Sample Introduction: A liquid biological sample containing a target antigen or antibody is introduced into the sample chamber (7), which contains magnetic particles coated with capture antibodies.

[0144] • Step B3: Immunocapture: The sample is transferred to reaction chamber (13) where target analytes bind to antibody-coated magnetic particles during incubation.

[0145] • Step B4: Magnetic Separation and Washing: Magnetic particles are immobilized using the magnet actuation module. Unbound sample components are transferred to the waste chamber. One or more wash buffers are introduced to remove nonspecific binders.

[0146] • Step B5: Addition of Lanthanide-Labelled Antibody: A lanthanide- labelled secondary antibody solution is introduced and incubated to form a labelled immunocomplex on the magnetic particles.

[0147] • Step B6: Washing and Transfer: Unbound secondary antibodies are removed by washing. The labelled magnetic particles are resuspended in a suitable buffer and transferred to detection well (18).

[0148] • Step B7: Time-Resolved Detection: The detection well is excited with ultraviolet or suitable excitation light. Emission is detected after a predefined delay to suppress background fluorescence. The detected signal intensity is correlated to the concentration of the target analyte.

[0149]

[0093] EMBODIMENT C

[0150] Step-by-step process for Chemiluminescent Immunoassay (CLIA / TCLIA) In another embodiment, the system is configured to perform chemiluminescent immunoassay.

[0151] • Step Cl: Cartridge Identification: A CLIA-configured cartridge is inserted and identified by the analyser.

[0152] • Step C2: Sample Introduction: A biological sample containing a target antigen is introduced into the sample chamber (7), which contains magnetic particles coated with primary antibodies.• Step C3: Immunocomplex Formation: The sample is transferred to reaction chamber (13) where target analytes bind to primary antibody- coated magnetic particles during incubation.

[0153] • Step C4: Magnetic Immobilization and Washing: Magnetic particles are immobilized using the magnet actuation module. Unbound sample components are transferred to the waste chamber. One or more wash buffers are introduced to remove nonspecific binders.

[0154] • Step C5: Addition of Enzyme-Labelled Antibody: An enzyme-labelled secondary antibody (e.g., HRP-conjugated antibody) is introduced and incubated to form an enzyme-labelled immunocomplex.

[0155] • Step C6: Washing: Excess secondary antibody is removed by washing while immobilizing the magnetic particles.

[0156] • Step C7: Chemiluminescent Reaction: A chemiluminescent substrate is introduced into the detection well. The enzyme catalyzes a reaction producing light emission proportional to the amount of target analyte. • Step C8: Signal Detection: The emitted light is detected by a photodetector without external excitation. The signal is processed by the data analysis module to generate qualitative or quantitative outputs.

[0157]

[0094] EXEMPLARY MULTIPLEX PCR EMBODIMENT:

[0158] In one exemplary embodiment, said bio-analyser system (100) is configured to perform multiplex real-time PCR analysis of up to sixteen detectable parameters using a PCR cartridge comprising four spatially separated reaction wells (18), each reaction well (18) being optically interrogated in four spectrally distinct fluorescence detection channels.

[0159] • The total number of detectable parameters is defined as:

[0160] Total detectable parameters = number of wells x number of fluorescence channels.

[0161] [4 reaction wells x 4 fluorescence channels = 16 detectable parameters] • Multiplexing is achieved using combined spatial separation (reaction wells) and spectral discrimination (distinct fluorophores).• The target detectable parameters may include:

[0162] o pathogen-specific sequences;

[0163] o mutation-specific sequences;

[0164] o internal amplification controls (IC);

[0165] o positive controls (PC); and

[0166] o negative controls (NC).

[0167] • The configuration of targets and controls may vary and does not limit the scope of the invention.

[0168]

[0095] VALIDATION AND DECISION LOGIC

[0169] In one embodiment, the bio-analyser (100) comprises a data processing and control module configured to execute automated validation, quality control assessment and result interpretation logic based on Internal Control (IC), Positive Control (PC), and Negative Control (NC) performance.

[0170] • Internal Control (IC) Evaluation:

[0171] The data processing module evaluates amplification performance of at least one internal control to verify successful sample processing, nucleic acid extraction (where applicable), reagent integrity, and amplification efficiency. Failure of the internal control to meet predefined acceptance criteria results in classification of the test run as invalid.

[0172] • Positive Control (PC) Evaluation:

[0173] The positive control is evaluated to confirm reagent functionality, thermal cycling performance, and fluorescence detection integrity. Failure of the positive control to satisfy predefined acceptance thresholds results in classification of the run as invalid or inconclusive.

[0174] • Negative Control (NC) Evaluation:

[0175] The negative control is assessed to detect contamination, carryover, or nonspecific amplification. Detection of signal exceeding a predefined rejection threshold in the negative control results in run invalidation or qualification according to predefined decision criteria.

[0176] • Integrated Decision Logic:The system reports analyte-specific results only when:

[0177] o the internal control satisfies acceptance criteria;

[0178] o the positive control satisfies acceptance criteria; and

[0179] o the negative control remains below its predefined rejection threshold. Based on automated evaluation, the data processing module assigns one or more diagnostic status indicators, including:

[0180] o Valid;

[0181] o Invalid;

[0182] o Inconclusive; and

[0183] o Repeat Required.

[0184] The system may generate automated qualitative and / or quantitative outputs, including amplification curves, cycle threshold (Cq) values, and interpreted diagnostic calls, in accordance with predefined interpretation algorithms. The validation logic, threshold criteria, and reporting format may be configured according to assay requirements and do not limit the scope of the invention.

[0185]

[0096] ADVANTAGES:

[0186] ■ The said integrated bio-analyser system (100) provides high efficiency, automation and analytical accuracy in diagnostic testing, making it suitable for use in clinical laboratories, research environments, point-of- care settings, and field diagnostics applications.

[0187] ■ The system integrates real-time PCR, TRFIA and Particle-based CLIA within a single multifunctional platform, thereby eliminating the need for separate machine and consumables.

[0188] ■ The system comprises an integrated optical detection assembly configured to perform real-time fluorescence detection for PCR amplification; time- resolved fluorescence measurement for TRFIA; and chemiluminescent signal detection without external excitation for CLIA.

[0189] ■ The system utilizes gravity-assisted fluid flow for sample movement through the plastic cartridge, which is controlled by valves.■ The system employs magnetic particles for binding and analysis of nucleic acids and proteins.

[0190] ■ The system is configured as a stand-alone single system for conducting all major analysis in a remote laboratory setting.

[0191] ■ The system further comprises an integrated battery backup module for uninterrupted work flow.

[0192]

[0097] The foregoing descriptions are merely specific embodiments of the present invention, but are not intended to limit the protection scope of the present invention. Any variation or replacement readily figured out by a person skilled in the art within the technical scope disclosed in the present invention shall fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

We claim,1. An automated multifunctional bio-analyser system (100) for detecting nucleic acids, proteins, antigens and antibodies, comprising,a plurality of microfluidic cartridges (102), each prefilled with reagents and buffers, configured to receive a sample (101) and perform sample processing, chemical and mechanical lysis, magnetic bead-based nucleic acid extraction, thermal cycling, immunochemical reactions, washing, and optical detection; andan analyser (103) configured to receive said cartridges (102) and automate sample movement, incubation, thermal cycling, optical readouts and magnetic bead manipulation, said analyser (103) comprising: o a thermal cycler module (76) for temperature-controlled reactions; o an optical module (62) for detecting fluorescent or chemiluminescent signals;o a magnet actuation module (79) for manipulating magnetic particles;o a sonication module (71) for mechanical sample disruption; and o a motor module (55) for actuating flow-control valves in said cartridge (102);wherein said plurality of cartridges (102) is a sealed, self-contained, air pressure-assisted gravity-flow microfluidic cartridge;wherein said modules are controlled by a program controller operatively linked to a data processing and analysis module (53) to enable precise and automated operation;wherein said system (100) is configured to perform real-time fluorescent, colorimetric, chemiluminescent analysis for detecting nucleic acid, protein, antigen and antibodies using Polymerase Chain Reaction (PCR), Time-Resolved Fluorescence Immunoassay (TRFIA), Fluorescence Immunoassay (FIA), and ChemiluminescentImmunoassay (CLIA), said analyser (103) is configured to perform one or more of said assays in a fully automated manner.

2. The system (100) as claimed in claim 1, wherein said cartridge (102) comprising,a sample chamber (8),a lysis or reaction chamber (13),one or more reagent chambers (9, 10, 11);one or more flow control valves (3, 4, 5);a waste chamber (14); andone or more optically transparent reaction wells (18);one or more vent channels (15, 27) for pressure equalization; and externally accessible grooves (6, 22, 28) for engagement with the analyser modules;wherein said cartridge (102) is configured to operate using air-pressure- assisted gravity-driven microfluidic flow.

3. The system (100) as claimed in claim 2, wherein said cartridge (102) further comprises:■ a piercing air chamber (37) having a spring-biased piercing pin (40) and a flip-lock mechanism (38) for secure and controlled operation; ■ a retainer block (45) configured to prevent piercing of sealed chambers during storage and transportation;■ fluidic channels (36) for interconnecting the chambers;■ air distribution channels (7, 15);■ a cartridge air cap (20) to assist pressure-driven fluid flow;■ an air reservoir chamber (33) to distribute pressurized air to selected chambers; and■ a piercing member (39) configured to release reagents.

4. The system (100) as claimed in claim 2, wherein said flow-control valves comprise direct through-hole valves (3, 5) and an angular valve (4).

5. The system (100) as claimed in claim 2, wherein said reagent chambers are sealed with aluminium foil.

6. The system (100) as claimed in claim 1, wherein the analyser (103) further comprises a cartridge holding mechanism (69) configured to position the cartridge (102) within the analyser, said cartridge holding mechanism (69) comprising a base support frame (65), alignment rails (66), and locking brackets (67) for repeatable positioning and engagement of engagement posts (96) and end-stop elements (97) of the cartridge (102).

7. The system (100) as claimed in claim 1, wherein said analyser (103) comprises a barcode module (70) configured to read QR code data printed on the cartridge (102), including assay type, thermal profile and process parameters.

8. The system (100) as claimed in claim 1, wherein the analyser (103) further comprises a linear stage assembly (74) configured to position the optical module (62) relative to the reaction wells (18).

9. The system (100) as claimed in claim 1, wherein said thermal cycler module (76) comprises:■ a thermally conductive block (93) having a plurality of recesses or contact surfaces corresponding to PCR reaction wells (18);■ a thermoelectric element (94) thermally coupled to the thermally conductive block (93);■ a heat dissipation assembly (95) comprising a heat sink and a cooling fan; and■ one or more temperature sensors (96) positioned adjacent to the thermally conductive block (93);wherein said thermal cycler module (76) is mechanically isolated from adjacent modules and thermally insulated.

10. The system (100) as claimed in claim 1, wherein said magnet actuation module (79) comprises a linear actuator (79A) and a magnet (79b) for capturing and releasing magnetic beads / particles in the cartridge (102).

11. The system (100) as claimed in claim 1, wherein said sonication module (71) comprises a sonicator probe (70f) and holder (70e) for mechanically disrupting sample material in the cartridge (102), and operating at 20-30 kHz in pulsed intervals for mechanical disruption.

12. The system (100) as claimed in claim 1, wherein said optical module (62) comprises a plurality of optical fibers (95) connected to a fiber holder (87), a detector module (90) for light detection, an excitation light source including UV and monochromatic LEDs, a lens assembly for collimating and focusing light, one or more filters for selecting excitation and emission wavelengths, and a linear stage (91) for positioning the optical assembly relative to said optical fibers (95).

13. The system (100) as claimed in claim 1, wherein said motor module (55) comprises one or more encoder-controlled stepper motors configured to actuate valves (3, 4, 5) in said cartridge (102) to control fluid flow.

14. The system (100) as claimed in claim 1, wherein the analyser (103) further comprises a data processing and analysis module (53) configured to:process optical signals;perform baseline correction and normalization;calculate Cq / CT values for amplification assays relative fluorescence units (RFU); and- validate assay results using internal control logic; andgenerate qualitative or quantitative diagnostic outputs.

15. The system (100) as claimed in claim 1, wherein said multiplex optical system (62) detects at least sixteen analytical parameters using four reaction wells and four optical channels, including target nucleic acids, positive, negative, and internal controls.

16. The system (100) as claimed in claim 1, wherein said system (100) is portable and includes battery backup.

17. The system (100) as claimed in claim 1, wherein said cartridge (102) is singleuse disposable cartridge formed from thermostable polymer material selected from polycarbonate (PC), polyethylene (PE), and acrylonitrile butadiene styrene (ABS).

18. The system (100) as claimed in claim 1, wherein said cartridge (102) comprises an optical portion formed from transparent polycarbonate material for reaction monitoring.

19. The system (100) as claimed in claim 1, wherein said cartridge (102) is prefilled with one or more of: lysis buffer, magnetic beads, primers, probes, master mix, substrates, antibody-coated particles; or labelled detection antibodies.

20. The system (100) as claimed in claim 2, wherein the lysis chamber (13) contains magnetic particles configured to bind nucleic acids or proteins.

21. The system (100) as claimed in claim 1, wherein said sample (101) may include blood, body fluids, animal samples, human samples, plant samples, food matrices, environmental samples, and forensic samples.

22. The system (100) as claimed in claim 1, wherein said cartridge (102) for PCR- based nucleic acid detection comprises:■ a sample chamber (8) containing chemical lysis solution (e.g., NaOH, GITC) and optional lyophilized magnetic or mechanical disruption beads;■ a lysis chamber (13) containing lyophilized Proteinase K, Poly A, and / or zirconium beads;■ a magnetic bead chamber (11) for nucleic acid capture;■ one or more reagent chambers (8, 9, 10) containing wash buffers, elution buffers, primers, probes, and master mix; and■ preloaded PCR reaction wells (18) containing assay-specific primers, probes, master mix, and internal controls;wherein said PCR master mix comprises PCR buffer, dNTPs, MgCh, DNA polymerase, primers, probes, and a lyostabilizer, and wherein baseline fluorescence, RFU, and Cq values are automatically calculated using the data processing module;wherein said cartridge (102) is sealed, single-use, and configured for automated air-pressure-assisted gravity flow.

23. The system (100) as claimed in claim 1, wherein said cartridge (102) for time- resolved fluorescence immunoassay comprises:■ a sample chamber (8) containing lyophilized antibody-coated magnetic beads;■ chambers (9, 10, 11) containing wash buffers, fluorescently labelled secondary antibodies, or dilution buffers; and■ detection wells (18) for time-resolved fluorescence measurement.

24. The system (100) as claimed in claim 1, wherein said cartridge (102) for chemiluminescent immunoassay (CLIA) comprises:■ a sample chamber (7) containing primary antibody-coated magnetic beads;■ chambers (8, 9, 10, 11) containing wash buffers, enzyme-labeled secondary antibodies, chemiluminescent substrates, or stop solutions; and■ detection wells (18) for chemiluminescent signal measurement.

25. A method for analysing a biological sample (101) using a cartridge (102) for PCR-based nucleic acid detection, in a bio-analyser system (100), comprising the steps of:Step (a): inserting an assay-specific PCR cartridge (102) into the analyser (103) of said bio-analyser system (100), and identifying, by a barcode module (70) of said analyser (103), a cartridge type and an associated predefined PCR workflow, and automatically initiating the predefined PCR workflow;Step (b): introducing the biological sample (101) into a sample chamber (7) of said cartridge (102), said sample chamber (7) containing a chemical lysis solution configured to lyse cells and / or viral particles to release nucleic acids;Step (c): transferring the biological sample (101) to a lysis chamber (13) by actuating one or more valves, and performing chemical lysis using the lysis solution therein, and mechanical lysis enhanced by sonication using the sonication module, and / or bead-assisted agitation using mechanical disruption beads present in the lysis chamber;Step (d): introducing magnetic beads into the lysis chamber (13) and incubating the magnetic beads with the lysed sample to bind released nucleic acids to surfaces of the magnetic beads;Step (e): actuating a magnet actuation module () to immobilize the magnetic beads within the lysis chamber (13), and directing bead-free lysate to a waste chamber (14);Step (f): sequentially introducing one or more wash buffers from one or more wash buffer chambers (9, 10) into the lysis chamber (13), immobilizing the magnetic beads after each wash using the magnet actuation module, and transferring wash liquids to the waste chamber (14);Step (g): introducing an elution buffer into the lysis chamber (13) to release nucleic acids from the magnetic beads, and transferring an eluted nucleic acid solution into one or more PCR reaction wells (18);Step (h): subjecting the PCR reaction wells (18) to thermal cycling using a thermal block assembly of the bio-analyser system (100), the thermal cycling comprising denaturation, annealing, and extension steps, wherein each PCR reaction well (18) contains a lyophilized master mix, one or more primers, one or more probes, and at least one internal control; andStep (i): performing real-time fluorescence detection during amplification by exciting and detecting fluorescence signals using a multiplex optical detection system, acquiring fluorescence data for each thermal cycle, and processing the fluorescence data to generate amplification curves and corresponding cycle threshold (Cq) values.

26. A method for analysing a biological sample (101) using a cartridge (102) for time-resolved fluorescence immunoassay (TRFIA) in a bio-analyser system (100), comprising the steps of:Step (a): inserting a TRFIA-configured cartridge (102) into an analyser (103) of the bio-analyser system (100), and identifying, by a barcode module of the analyser (103), a cartridge type and an associated predefined immunoassay workflow, and automatically initiating the predefined workflow;Step (b): introducing a liquid biological sample (101) comprising a target antigen or antibody into a sample chamber (7) of the cartridge (102), the sample chamber (7) containing magnetic particles coated with capture antibodies;Step (c): transferring the biological sample (101) to a reaction chamber (13) by actuating one or more valves, and incubating the sample with the magnetic particles to permit binding of target analytes to the capture antibodies to form antigen-antibody complexes on surfaces of the magnetic particles;Step (d): actuating a magnet actuation module of the bio-analyser system (100) to immobilize the magnetic particles within the reaction chamber (13), transferring unbound sample components to a waste chamber, and introducing one or more wash buffers to remove non-specifically bound components;Step (e): introducing a lanthanide-labelled secondary antibody solution into the reaction chamber (13), and incubating to allow formation of a labelled immunocomplex on the magnetic particles;Step (f): immobilizing the magnetic particles using the magnet actuation module, removing unbound secondary antibodies by one or more washing steps, resuspending the labelled magnetic particles in a buffer solution, and transferring the resuspended particles to at least one detection well (18); andStep (g): exciting contents of the detection well (18) using ultraviolet or other suitable excitation light, detecting time-resolved fluorescence emission after a predefined delay interval to suppress background fluorescence, and correlating a detected signal intensity to a concentration of the target analyte present in the biological sample (101).

27. A method for analysing a biological sample (101) using a cartridge (102) for chemiluminescent immunoassay (CLIA) in a bio-analyser system (100), comprising the steps of:Step (a): inserting a CLIA-configured cartridge (102) into an analyser (103) of the bio-analyser system (100), and identifying, by a barcode module of the analyser (103), a cartridge type and an associated predefined chemiluminescent immunoassay workflow, and automatically initiating the predefined workflow;Step (b): introducing a biological sample (101) comprising a target antigen into a sample chamber (7) of the cartridge (102), the sample chamber (7) containing magnetic particles coated with primary antibodies specific to the target antigen;Step (c): transferring the biological sample (101) to a reaction chamber (13) by actuating one or more valves, and incubating the sample with the magnetic particles to permit binding of the target antigen to the primary antibodies to form immunocomplexes on surfaces of the magnetic particles;Step (d): actuating a magnet actuation module of the bio-analyser system (100) to immobilize the magnetic particles within the reaction chamber (13), transferring unbound sample components to a waste chamber, and introducing one or more wash buffers to remove non-specifically bound components;Step (e): introducing an enzyme-labelled secondary antibody into the reaction chamber (13), the enzyme-labelled secondary antibody comprising an enzyme conjugate configured to bind to the target antigen or to the primary antibody, and incubating to form an enzyme-labelled immunocomplex on the magnetic particles;Step (f): immobilizing the magnetic particles using the magnet actuation module and removing excess enzyme-labelled secondary antibody by one or more washing steps;Step (g): introducing a chemiluminescent substrate into at least one detection well (18) containing the magnetic particles, wherein the enzyme catalyzes a reaction of the chemiluminescent substrate to generate lightemission proportional to an amount of the target antigen present in the biological sample (101); andStep (h): detecting the emitted light using a photodetector of the bio-analyser system (100) without external optical excitation, and processing a detected signal using a data analysis module to generate a qualitative and / or quantitative output indicative of the presence or concentration of the target antigen.

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