Single bead capacitive detector for microbial applications

Abstract

The present invention provides an improved capacitive bead sensor for detecting and / or quantifying a target analyte in a sample wherein the detection limit is as low as a single bead, the capacitive bead sensor can be repeatedly used for multiple bead testing or continuous bead flow, and the capacitive bead sensor is readily manufacturable and automatable. By using various structural advantages and electronic signal amplification techniques, the capacitive bead sensor enables sensitivity as low as single molecule detection without the need for enzymatic amplification such as PCR, these various structural advantages and electronic signal amplification techniques further allow for multiplex target detection not only across various nucleic acid targets but across the entire target class, thereby allowing for simultaneous detection of, for example, viral nucleic acids and host antibodies against these viruses.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 63 / 304,312, filed on January 28, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to the capture of nucleic acids and antibody / antigen molecules by magnetic beads, and methods of detecting / quantifying these molecules, down to the level of single beads and thus single molecules. Background Art

[0004] Capturing and detecting nucleic acids (NA) from biological samples has become a key aspect of early disease detection and monitoring. In conventional laboratory clinical assessments, molecular tests and serological (antibody / antigen) tests are used together to provide effective diagnosis. Ideally, such tests will be completed together from a sample. For example, PCR or LAMP tests can provide qualitative or possible semi-quantitative information about the presence of viral RNA, and separate antibody detection can determine a person's previous infection status. Quantification of captured NA is important, for example, in determining the viral load of infectious pathogens or in gene expression analysis for multiple captured NAs. Multiple testing, variant identification and genotyping have also become very important, as seen in the recent COVID19 epidemic, where 'variants of concern' (e.g., δ, Omicron) cause huge health problems and many deaths. Direct single molecule detection with ultra-high sensitivity can allow analysis of rare mutations that may cause cancer, detection of trace contamination of biopharmaceutical products by pathogenic DNA or RNA, very early or late detection of viral infection in patients with fairly low viral loads (see 'single-molecule-PCR' US5811235, which is incorporated herein by reference), and for cloning and template generation in DNA and RNA sequencing. The disadvantages of ultra-sensitive methods can be cross-contamination of amplicons, casual contact of suspects with crime scenes, and patients testing positive for the presence of pathogenic RNA or DNA long after they have recovered from the infection and are no longer contagious. These are specific features of enzymatic amplification methods.

[0005] These critical health diagnostics are currently only available in highly specialized laboratories, requiring highly trained staff and extensive equipment and infrastructure, all of which result in significant expenses.

[0006] The use of magnetic beads for nucleic acid capture is known. For this case, the optimal magnetic bead diameter range is 400nm to 2800nm. Non-magnetic particles (e.g., glass, silica, polystyrene, titanium dioxide, silver or gold) are also used for nucleic acid capture, and their diameters are even smaller, typically as low as 40nm. The notable use of nanoparticles in diagnostics is in lateral flow tests, where these nanoparticles are conjugated to antibodies for colorimetric detection of antigens, or vice versa, to antigens for antibody tests using the same technology.

[0007] The captured DNA is eluted and, if target DNA is present and hybridized to matching oligomer probes and primers, it is typically amplified exponentially in a polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) assay for optical detection. If the captured target is RNA, a reverse transcription step (RNA to cDNA) is first employed.

[0008] Conventional enzymatic amplification is used to detect the target NA (if present). However, due to the stochastic nature of exponential amplification, it is difficult to accurately quantify the target NA in a sample using these PCR and LAMP methods, and samples with low concentrations remain difficult to measure and quantify. Single molecule detection using PCR and LAMP is also difficult due to sample-specific enzymatic inhibition and the length of primer probes, which are typically >18 (usually 20+) and >50 nucleotides in length, respectively. This can prevent binding to key mutations such as mutations in the receptor binding domain (RBD) of the spike gene of the SARS-CoV-2 virus, where many single-base mutations (e.g., in the δ or Omicron variants) can occur within 15 to 20 nucleotides of each other. Droplet / digital PCR and sequencing can address single molecule detection and identification, but these methods typically require large and fairly expensive laboratory instruments and trained professional operators.

[0009] It has been proposed to detect magnetic beads carrying target molecules on complementary metal oxide semiconductor (CMOS) chips, such as Hall sensors, as described in Florescu et al. (2010) "On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis", Journal of Applied Physics (J Appl. Phys.) 107(5):054702, and GMR-SV sensors, as described in Murmann et al. (2013) "A 256 pixel magnetoresistive biosensor microarray in 0.18 μm CMOS", IEEE J Solid-State Circuits 48(5):1290-1301, the contents of each of which are incorporated herein by reference in their entirety.

[0010] However, each of these CMOS chips requires additional special layers of magnetics and metals, which are expensive and generally not available in standard high-volume CMOS foundry processes.

[0011] Chang and Lu (2013) “CMOS capacitive biosensors for highly sensitive biosensing applications”, Annual Int Conf IEEE Eng Med Biol Soc. 2013:4102-5, which is incorporated herein by reference in its entirety, proposed capacitive detection of magnetic beads on a CMOS chip.

[0012] Detection of non-magnetic particles (such as glass, silica, titanium dioxide, silver, gold, nanoparticles) usually requires expensive optical detectors, lasers, X-rays, or SEM or mass spectrometry instruments. In some lateral flow tests, visual detection with the human eye is possible, in which a large number of nanoparticles, typically 40 nm, are captured at the detection (or control line), enabling visual detection. But the sensitivity of these methods is limited to thousands of particles.

[0013] U.S. Pat. No. 10,746,683 (Cummins et al.), the contents of which are incorporated herein by reference in their entirety, describes an interdigitated electrode (IDE) capacitive sensor for detecting magnetic and nonmagnetic particles as listed above. This is a disposable sensor with a lower limit of detection of 200 beads, where the water or buffer carrier evaporates, leaving the beads permanently attached to the sensor surface.

[0014] U.S. Patent No. 10,160,966 (O'Farrell et al.), the contents of which are incorporated herein by reference in their entirety, discloses beads that can be provided to a sample, and peptide-nucleic acid (PNA) probes attached to the beads that hybridize to and capture target nucleic acid molecules. This is a one-time arrangement.

[0015] US11,459,601 (O'Farrell et al.), the contents of which are incorporated herein by reference in their entirety, discloses various assay steps for nucleic acid detection. Paramagnetic transport (T) beads attached to a first PNA probe and captured nucleic acid are magnetically removed from the sample and moved through various washing and tethering steps. Reporter (R) beads attached to a second PNA probe are then tethered to the captured target nucleic acid (if present), resulting in a target-specific sandwich assay. The beads are then moved to a wash chamber and onto a CMOS sensor chip, where the R beads are eluted and detected. This is also a disposable arrangement. Summary of the invention

[0016] The systems and methods of the present disclosure generally relate to the detection and / or quantification of target analytes in samples. Through the various structures and techniques discussed herein, alone or in combination, sensitivity as low as single molecule detection can be obtained without enzymatic amplification (such as PCR). The systems and methods of the present invention can use various structural advantages and electronic signal amplification technologies, which further allow multiple target detection not only across various nucleic acid targets but also across entire target categories, thereby allowing simultaneous detection of, for example, viral nucleic acids and host antibodies against these viruses.

[0017] Aspects of the invention may include methods for nucleic acid detection, the methods comprising detecting a target nucleic acid present in a sample at 100 copies / mL or less using a capacitive sensor and without enzymatic amplification. In various embodiments, depending on the desired sensitivity, accuracy, and complexity of the assay, the techniques discussed throughout may be used alone or in combination to achieve detection of 150 copies / mL or less, 250 copies / mL or less, 500 copies / mL or less, 1000 copies / mL or less, 5000 copies / mL or less, 10,000 copies / mL or less, 50,000 copies / mL or less, 100,000 copies / mL or less, 1,000,000 copies / mL or less, etc. In certain embodiments, the methods may be operated to achieve at least 10 copies / mL in the detection of the target nucleic acid. 9 In various embodiments, again depending on the assay parameters, the method can achieve at least 10 8 times, at least 10 7 times, at least 10 6 times, at least 10 5 times, at least 10 4 times, at least 10 3 times or at least 10 2 Similarly, the method can be operated to achieve signal amplification equivalent to at least 30 PCR cycles, at least 25 PCR cycles, at least 20 PCR cycles, at least 15 PCR cycles, at least 10 PCR cycles, or at least 5 PCR cycles in the detection of the target nucleic acid.

[0018] The full scale range of nucleic acid detection performed by a capacitive sensor can be at least about 8pF, at least about 7pF, at least about 6pF, at least about 5pF, at least about 4pF, at least about 3pF, at least about 2pF, or at least about 1pF. The method can include using a capacitance digital converter to convert the signal from the capacitance sensor. The capacitance digital converter can be a ∑-Δ 24-bit capacitance digital converter. The nucleic acid detection performed by the capacitance sensor can include a resolution of at least about 0.5aF and an accuracy of at least about 4aF. In various embodiments, by including an internal offset capacitor in the reference input, the full scale range of nucleic acid detection performed by the capacitance sensor can be at least about 15pF.

[0019] The method of the present invention may further include binding the target nucleic acid to a reporter (R) bead; passing the R bead through a sensor region comprising two capacitive electrodes located on a substrate and connected to a signal processing circuit; and detecting the R bead using the signal processing circuit as the R bead passes through the gap. The two electrodes may be spaced apart to form a gap so that only a single bead moves between the gap at a time. The two electrodes may form a groove through which the R bead passes. The method may further include passing the R bead through multiple sensor regions; and detecting the R bead as the R bead passes through the multiple sensor regions by applying one or more of a maximum likelihood estimation (MLE) machine learning algorithm, a partial response maximum likelihood (PRML) algorithm, or a Viterbi algorithm to the signals received by the signal processing circuit from the multiple sensor regions.

[0020] In some embodiments, detecting the target nucleic acid may include combining the target nucleic acid with a reporter (R) bead, and detecting the R bead using a capacitive sensor. Detecting the R bead using the capacitive sensor may include detecting the R bead flowing through the sensor in a fluid. The fluid may be an oil. The oil may be a silicone oil. The fluid may include a fluorinated carbon. The fluid may be selected from dodecafluoro-2-methylpentan-3-one and methoxy-nonafluorobutane. The method may include fluorinating the R bead. The method may include forming a water layer around the R bead flowing in the fluid to amplify the detection signal at the capacitive sensor.

[0021] In certain embodiments, the method may include binding the target nucleic acid to the R beads using a bead-bound peptide nucleic acid (PNA). The bead-bound PNA may include a ligand or linker PNA that does not interact with the target RNA. The method may include detecting a plurality of different targets using one or more capacitive sensors, the plurality of different targets including at least the target nucleic acid present in the sample. The plurality of different targets may include a plurality of different target nucleic acids. The plurality of different target nucleic acids may be derived from different pathogens or different variants of pathogens. The pathogen may be SARS-CoV-2. The plurality of different targets may include a protein. The protein may be an antibody. The protein may be an antigen. In some embodiments, the target nucleic acid may be derived from a dengue virus, and the antibody may be a dengue-specific antibody. In certain embodiments, the target can be a drug, including but not limited to Onpattro, Patisiran, givosiran, lumasiran, and inclisiran (siRNA) or an antibody cocktail, such as REGEN-COV (casirivimab and imdevimab).

[0022] The target can be an mRNA vaccine, such as tozinameran. Co-monitoring of therapeutic agents and viral load is advantageous, and the multiple properties of the methods and systems of the present invention can allow such co-monitoring. The stability of some RNA-derived drugs before and after use is a matter of concern, and the technology of monitoring the concentration or level of these drugs from blood or saliva samples, for example, before injection and after use, is valuable. In pharmaceutical companies, the same technology can be used for QC of these technologies. The target can include target nucleic acid derivatives, which can be synthetic and include non-standard bases, such as Nl-methyl pseudouridine. The target can also be a phospholipid or glycolipid. The target can be located on the surface of a cell, a viral particle, or a lipid nanoparticle, wherein the cell, viral particle, or lipid nanoparticle allows tethering.

[0023] Detecting the plurality of different targets may include binding each of the plurality of targets to a different bead; and detecting each of the different beads using the capacitive sensor. The method may further include distinguishing the different beads based on different capacitive detection signals from the capacitive sensor. In some embodiments, the method may include selectively releasing the R beads from the substrate-bound targets based on the different sensor-bound targets to which the R beads are bound; and detecting the effect of releasing the R beads on capacitance.

[0024] Detecting the multiple different targets may include binding each of the multiple different targets to a different probe on a substrate. The substrate may be operably associated with one or more capacitive sensors. The method may further include binding one or more reporter (R) beads to each of the substrate-bound targets of different substrate-bound targets. In some embodiments, the method may include selectively releasing the R beads from the substrate-bound targets based on the different substrate-bound targets to which the R beads are bound; allowing the released R beads to flow through the capacitive sensor in a fluid; and detecting the released R beads flowing through the sensor. Each of the different beads may have a different size, and the method may include microfluidically guiding each bead to a different capacitive sensor based on the size of each of the different beads. The guiding step may include one or more of an inertial method, a dielectrophoresis method, or a magnetoelectrophoresis method.

[0025] Detecting the target nucleic acid may further include binding the target nucleic acid to a probe on a substrate; and binding a plurality of reporter (R) beads to the substrate-bound target nucleic acid. The plurality of R beads may bind to different sequences in the substrate-bound target nucleic acid. The substrate may be operably associated with the capacitive sensor. The method may further include releasing the plurality of R beads from the substrate-bound target nucleic acid; allowing the released R beads to flow through the capacitive sensor in a fluid; and detecting the released R beads flowing through the sensor.

[0026] In certain embodiments, detecting the target nucleic acid can include binding transport (T) beads to the target nucleic acid in the sample to form a T-bead complex; binding the T-bead complex to reporter (R) beads to form an R-bead complex; eluting the R beads and the T beads from the R-bead complex; detecting the R beads using a capacitive sensor; and returning the T beads to the sample to bind to another target nucleic acid.

[0027] Aspects of the present invention may also include systems and architectures as described herein, which are operable to perform any of the methods described above.

[0028] Aspects of the present invention can include a system for nucleic acid detection, the system includes a capacitive sensor, the capacitive sensor further includes a signal processing circuit and a sensor area, the sensor area includes two capacitive electrodes on a substrate that are connected to the signal processing circuit, wherein the two electrodes are spaced apart to form a gap, and wherein only a single bead moves between the gap at any time, and the electrodes detect the bead and / or any molecule connected to the beads. The system can be reusable after being used to detect beads. The capacitive sensor can be about 2mm x2mm. The bead or particle can be contained in water, buffer, or oil, or fluorocarbon or an organic solvent. The bead or particle detected can be a substitute for a microbial analyte determined from upstream. The bead or particle can be magnetic or nonmagnetic.

[0029] The gap can be twice the diameter of the bead. In some embodiments, the substrate can be 3D printed. The electrodes can be formed by sputtering or ink deposition and patterning. The spacing between the electrodes can be about 0.8 μm to 20 μm. The substrate can be a CMOS semiconductor chip. The electrodes can be etched in a metal layer, and the spacing between the electrodes can be between about 40 nm and about 5 μm. In certain embodiments, the systems and methods of the present invention may further include one or more of ultrasonic oscillation, dielectrophoretic bead steering, oil injection, and / or regulating the zeta potential of the beads to prevent bead agglomeration and / or induce movement of particles and beads through the system.

[0030] The purpose of the present invention is to make key health diagnostics available in a simple portable form outside the laboratory, requiring little or no operator training, for use at the point of care and home. However, the same technology can reduce the size, scale, power consumption and logistical costs of the central laboratory. Another purpose of the present invention is to be able to detect nucleic acids and antibodies from patient samples simultaneously and quickly. This simultaneous detection may be advantageous for situations such as dengue outbreaks, in which case it is crucial not only to detect and distinguish dengue virus (DENV) RNA variants 1, 2, 3, 4 (indicating current active infection), but also to detect and identify DENV antibodies. When classifying, it may be helpful to determine whether the patient has a history of dengue infection before making any clinical decision about the application of dengue vaccine.

[0031] The systems and methods described herein recognize and address certain shortcomings in the technologies discussed above. For example, Florescu's beads are quite large, typically 2.8 μm or larger, and the beads need to be precisely manipulated onto a circular Hall sensor with a diameter of 4.5 μm. This fine manipulation of individual particles is impractical in automated commercial NA assays. Murmann's magnetic particles are smaller (50 nm), but the detection limit is 2000 particles, making it unsuitable for most nucleic acid tests and gene expression analysis. The diameter of the beads described in Chang and Lu is 10 μm, which is too large for most molecular analysis assays, and the beads need to be electromagnetically manipulated into the precise center of the coil. This also makes it impractical for commercial or automated assays. In addition, the sensing mechanism described in Chang and Lu is not capacitive, because the capacitance of Chang and Lu actually decreases in the presence of beads. Therefore, the demand for a reusable sensor that can detect a continuous bead stream and detect individual beads is not met. In addition, the demand for a continuous bead stream to achieve single molecule analysis is not met. There is an unmet need for continuous T-bead and R-bead flow to enable single molecule analysis, genotyping and variant identification, as well as for simple portable instruments and methods for nucleic acid detection, variant identification, viral load quantification and single molecule detection analysis in non-laboratory settings (such as point-of-care and community clinics) to allow rapid clinical intervention, treatment and real-time monitoring in epidemic outbreak situations. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Depicted is an analyte droplet with beads on a sputtered ITO electrode on a PET film.

[0033] Figure 2 The capacitance-to-digital converter and the connection socket to the PET film are depicted.

[0034] Figure 3 Depicted is a 24-bit sigma-delta capacitance-to-digital converter architecture and circuit.

[0035] Figure 4 Depicted is an ink-printed laser-ablated electrode capacitor on a 3D-printed substrate.

[0036] Figure 5 yes Figure 4 Side view photograph of the ink-printed laser-ablated electrode.

[0037] Figure 6 Depicted is an etched aluminum electrode IDE capacitor on a CMOS silicon chip.

[0038] Figure 7 Depicted is the formation of a CMOS semiconductor with aluminum and copper electrodes.

[0039] FIG. 8 depicts a prior art analyte sensor with tethered beads and its equivalent circuit.

[0040] Fig. 9 is Figure 6 Photograph of beads specifically captured on a circular CMOS sensor.

[0041] Fig.10 The end-to-end assay performance is depicted in a graph of capacitance versus RNA.

[0042] Fig.11 Depicted is the signal amplification achieved by specific tethering of many beads to one sensor.

[0043] Fig.12 Depicted is a PNA probe targeting a conserved region of the SARS-CoV-2 virus.

[0044] Fig.13 and Fig.14 Additional PNA probes targeting regions of the SARS-CoV-2 virus are depicted.

[0045] Fig.15 Antigenic peptide sequences of dengue envelope and NS1 proteins are depicted.

[0046] Fig.16 and Fig.17 PNA probes targeting dengue virus variants 1, 2, 3, 4 are depicted.

[0047] Fig.18 Thirty CMOS sensors are depicted with PNA probes spotted on some of the sensors.

[0048] Fig.19 Depicted is the spotting pattern of the sensor for dengue RNA and antibody detection.

[0049] Fig. 20 Depicted are dengue RNA and antibody assays side-by-side on a CMOS sensor chip.

[0050] Fig.21 Depicts the rows of ground on a CMOS silicon chip Figure 6 Several IDE capacitors in the IDE capacitors.

[0051] Fig. 22 Some sensors have clear silicone oil Fig.21 Photo of the chip.

[0052] Fig.23 yes Fig.21 Schematic diagram of a cylinder of oil above one of the circular sensors.

[0053] Fig.24 Depicted Fig.23 C 硅酮油 and C 氮化物 The equivalent circuit of .

[0054] Fig.25 yes Fig.21 Photograph of a bead containing PD5 oil film on a circular sensor.

[0055] Fig.26 Is a circular sensor with Fig.25 Schematic diagram of a cylinder of beads.

[0056] Fig. 27 Depicted Fig.26 C 硅酮油 , C 氮化物 , C 珠粒 The equivalent circuit of .

[0057] Fig.28 Sensor measurements of PD5 oil capacitance with and without beads are provided.

[0058] Fig.29 Depicted are 0.4 μm beads in 0.8 μm trenches between metal electrodes.

[0059] Fig.30 is the electric field simulation of this bead passing between electrodes in oil.

[0060] Fig.31 Depicted are the calculated capacitance of the beads as they pass between electrodes.

[0061] Fig.32 Depicted in Fig.30 and 31 The boundary element model used in the simulation.

[0062] Fig.33 Describes exactly Fig.29 The ground plane underneath the electrode.

[0063] Fig.34 Shows Fig.33 The capacitance of the bead is calculated with the ground plane located underneath.

[0064] Fig.35 Shown is the real-time capacitance of a series of beads flowing between electrodes.

[0065] Fig.36 Beads are depicted flowing between multiple electrodes and then down the holes in the substrate.

[0066] Fig.37 Depicted are laser-formed vias in the ink electrode and 3D-printed substrate.

[0067] Fig.38 Depicted is an ink-printed electrode with a narrow gap formed by laser ablation.

[0068] Fig.39 Depicted is an assay with recycling of T beads according to certain embodiments.

[0069] Fig.40 Depicted are embodiments enabling single molecule analysis of, for example, SARS-CoV-2.

[0070] Fig.41 An at-home breath saliva self-testing embodiment for COVID / flu / hay fever is depicted.

[0071] Fig.42 Fluorinated reporter beads flowing through a fluid phase change are demonstrated.

[0072] Fig.43 A microfluidic embodiment for flowing beads through a phase change is demonstrated.

[0073] Fig.44 R beads are depicted tethered to D beads providing additional amplification.

[0074] Fig.45 The R-bead-D-bead tethered complex is shown flowing between sensors.

[0075] FIG. 46 shows a RD bead complex tethered to (a) a CMOS sensor, (b) a bead substrate.

[0076] Fig.47 Shown are R nanoparticles of reduced size (1-100 nm) tethering D beads to the surface.

[0077] Fig.48R nanoparticles with linker-type molecular shapes are shown.

[0078] Fig.49 The linker is shown to be a ligand that does not interact with the target RNA.

[0079] Fig.50 R nanoparticles are shown tethering D beads to the surface of a magnetic particle. DETAILED DESCRIPTION

[0080] The present invention provides an improved capacitive bead sensor that can be reused for multiple bead tests or continuous bead flows, and the capacitive bead sensor is easily manufacturable and automatable. The systems and methods described herein allow detection down to individual beads and thus single target molecules. By electronic and other signal amplification techniques discussed below, this sensitivity can be achieved without enzymatic amplification, thereby avoiding some of the shortcomings of those enzymatic methods as described above. In addition, the systems and methods described herein can achieve the desired sensitivity while maintaining a wide dynamic range that is particularly useful in multiplex analysis of target molecules of different concentrations in a sample.

[0081] As discussed in more detail below, the systems and methods described herein can enable, among other things, one or more of the following: a) simultaneous capture and detection of nucleic acids, antibodies, and antigen molecules from a biological sample; b) quantification of these molecules down to single-digit copy levels (e.g., for clinical viral load determination); c) gene expression analysis by simultaneous quantification of multiple PNA-captured genes on multiple capacitive bead sensors; d) single molecule detection analysis by multiple PNA probes on multiple beads that are recycled through sample and assay steps; and e) distinguishing current from past infection by simultaneously detecting both RNA and antibodies from the same sample.

[0082] Bead detection and signal amplification

[0083] Figure 1Six sputtered indium tin oxide (ITO) capacitive electrode sensors on a polyethylene terephthalate (PET) film with a thickness of 150 μm are depicted. Each capacitor is approximately 2 mm x 2 mm. A connector socket (on the right) connects the capacitor to a signal processor, which may contain a capacitance-to-digital converter, analog and digital filters, non-volatile memory, a signal processor, and wireless communications (Bluetooth, WiFi, GSM, GPRS, etc.). The capacitances shown (4.45 pF, 4.587 pF, 4.59 pF) are baseline values ​​with no liquid or beads. The slight increase in values ​​(S1-S3-S5) reflects the longer wires reaching each capacitor. These baseline values ​​can be stored as offsets in the non-volatile memory of the signal processing circuit to zero all sensors to a zero baseline (e.g., by subtracting the offset from each of the associated sensor readings). Fig.28 ). The sensors are arranged in a linear row to facilitate integration with a droplet dispensing tip for assay automation in some applications.

[0084] Figure 2 Depicts capacitance-to-digital converters on a printed circuit board (PCB) and connecting these capacitance-to-digital converters to Figure 1 The six capacitors have miniature sockets.

[0085] Figure 3 The circuit architecture of a sigma-delta capacitance-to-digital converter as known in the art (e.g., "Oversampled Delta-Sigma Data Converter" (Candy et al., Wiley-IEEE Press, 1992)) is depicted. Using standard switched capacitor technology, operating at a modulator clock frequency in the KHz to MHz range, C 传感器 With C 参考 The difference is amplified and integrated and then applied to the comparator. The comparator output is fed back to control V 参考 The switch is actually a 1-bit digital-to-analog converter (DAC). The comparator bitstream output is applied to a decimation filter to produce a digital result with up to 24-bit resolution. Typical conversion times are 1mS to 100mS. Million-fold electronic amplification provided by multi-stage integrated amplifiers can contribute to the overall measurement amplification.

[0086] Figure 4Depicted is a 3D printed polylactic acid (PLA) substrate with interdigitated electrodes patterned using ink printing and laser ablation. The measured baseline capacitance (including the fingers and wiring parasitics) is in the region of 4.5pF to 6.1pF. The fringe field sensing or transduction portion of the capacitance can be in the region of 0.4pF to 1.6pF, depending on the electrode spacing, which can typically be 0.8μm to 20μm, depending on the spot size of the ablation laser.

[0087] Figure 5 yes Figure 4 A side view of the ink electrodes of the S4 in Figure 1. Laser ablation of the ink to create the electrode structure forms trenches between the electrodes, where the height of the ink thickness can be programmed from a few microns to hundreds of microns during 3D printing. Electrohydrodynamic inkjet printing is another method to create fine-line (e.g., 1 μm) electrode features without laser ablation.

[0088] Figure 6 Depicted is an interdigitated electrode (IDE) capacitor formed by subtractive etching of the top metal layer of a complementary metal oxide semiconductor (CMOS) chip. In this embodiment, a sigma-delta capacitance-to-digital converter can be formed in the same CMOS chip located below or adjacent to the sensor. This significantly reduces parasitic capacitance and noise (thermal and electromagnetic), thereby providing a wider dynamic measurement range.

[0089] Figure 7 A cross-sectional view of the metal layers forming a CMOS sensor is shown in (an illustrative example from Burghartz et al., IEEE 2004, DOI 10.1109 / TED.2004.823325). This reference also describes typical CMOS process formation steps and is incorporated herein by reference in its entirety. Modern dry plasma etching and highly directional anisotropic deep reactive ion etching (DRIE) methods used in semiconductor manufacturing can achieve electrode spacings as low as 20 nm. In some embodiments, Figure 7 The dashed lines in (a) show narrow spaces in the top metal that can be used as grooves between which beads flow without passivation. This can be advantageous in eliminating manufacturing steps (passivation) to achieve narrower spacing while providing increased assay amplification. When covered in silicone oil where beads can flow, the metal does not oxidize or corrode. Figure 7 The dashed line (b) in Figure 1 is a variation of copper plating for much thicker metal structures, similar to Figure 53D printed trenches. This copper plating example of Burghartz is used for RF inductor coil formation. In certain embodiments, the system of the present invention can use this standard high volume RF-CMOS thick metal technology as the electrode trench structure for measuring beads in the trench and measuring the flow of beads in oil. This can avoid the requirement for special CMOS layers or non-standard semiconductor processing.

[0090] FIG8 shows a capacitive bead detection method (see U.S. Patent No. 10,746,683, which is incorporated herein by reference in its entirety). The bead can simply arrive at the sensor surface (8b), or be tethered specifically to the sensor surface (8c), for example, by probe 0 and tethered by an additional probe 1 connected to the bead. The capacitive fringe field (dashed line of the electric field in 8b) forms a transducer that detects C due to the dielectric constant of the bead material. 珠粒 8(a) shows the equivalent circuit. 氮化物 With C 珠粒 Series connection occurs because the field lines pass through the insulating nitride protection layer.

[0091] Fig. 9 FIG8(c) is a photographic illustration of tethering, in this case showing a PNA probe designed to target the HIV gag gene in Figure 6 Since these beads are surrogate beads for HIV-RNA in the upstream assay from the patient sample, quantification of sensor capacitance as the number of beads is also a direct digital readout of HIV viral load in the patient sample. Fig.10 The end-to-end assay performance of "Capacitance vs. RNA" is illustrated in the figure. The top photo shows beads specifically captured on the sensor surface. The bottom photo shows that beads are not captured when an off-target PNA probe is used.

[0092] Fig.11 A target RNA strand is shown tethered to the sensor surface (via probe 0), with several beads tethered to the RNA via probes 1, 2, 3, 4, etc. These probes can be designed to target different conserved regions of the target DNA or RNA, giving very high specificity of target capture. This also results in significant signal amplification due to the increased number of captured beads tethered to the sensor. This simulates dendritic amplification and can contribute to overall assay amplification so that DNA or RNA can be quantified even at very low copy number levels without the need for enzymatic amplification.

[0093] Peptide and probe design for nucleic acid and antibody assays

[0094] Fig. 9The sequence of the HIV Gag gene PNA probe has been published by Zhao et al., Nature Communications (nComms 5079), the contents of which are incorporated herein in their entirety. Fully synthetic PNA probes can be chemically synthesized using solid phase peptide synthesis (SPPS) by FMOC chemistry.

[0095] Fig.12 A 14-base PNA probe targeting a unique region of the SARS-CoV-2 viral genome is shown, which is conserved across the Wuhan reference sequence and the α, β, γ, δ, and οBA1 and BA.2 variants. For example, the probe can be tethered to a sensor surface, such as Fig.11 Probe 0.

[0096] Fig.13 and 14 Additional PNA probes (probe 6, probe 7) designed to target the SARS-CoV-2 receptor binding domain (RBD) region are shown for distinguishing the oBA.1 and BA.2 variants from the a, β, γ, and δ variants. Probe 8 further narrows down and identifies the BA.2 variant by targeting the S gene discard deletion of the BA.2 variant. As is known in the art, additional probes are similarly applied to identify other variants, where variant identification and viral genotyping can be achieved with a small number of carefully designed probe sequences (e.g., by applying Fig.41 For example, in 2004 "A universal microarray for detection of SARS coronavirus" (doi:10.1016 / j.jviromet.2004.06.016), Long et al. published 20 probe sequences that "not only detect SARS-CoV but also identify the genotypes of six mutated bases related to the different phases of the SARS epidemic" (2003 / 2004).

[0097] Fig.15Synthetic peptide sequences of the envelope (E) and non-structural (NS-1) proteins of dengue virus (DENV) are shown (from Nagar et al., DOI 10.1155 / 2020 / 1820325). These sequences can also be chemically synthesized by FMOC SPPS. These sequences are based on linear epitopes of DENV envelope and non-structural proteins for testing and diagnosing dengue-specific IgM and IgG antibodies in patient antisera. The nine sequences shown are the "most antigenic / reactive" for high-specific antibody detection (from Nagar et al.).

[0098] Fig.16 and 17 An example of achieving 100% detection of four dengue reference genomes NC_001477, NC_001474, NC_001475, NC_002640 (DENV 1, 2, 3, 4, respectively) is shown, where only two PNA probes (14bp and 15bp) target the conserved regions shown. This is not possible for longer PCR probes and primers, which are usually >25nt and will therefore span some of the mutations shown, making specificity less than 100% and capture efficiency also low. The high capture efficiency of PNA can also contribute to overall assay signal amplification, and therefore contribute to sensitivity.

[0099] Multiple Testing

[0100] Fig.18 30 circular CMOS sensors (eg Figure 6 ) in which PNA-tethered probes are spotted onto individual sensors. These probes can specifically capture reporter R beads, such as Fig. 9 The beads seen in the circle and Fig.11 Thirty sensors enable a higher order of multiplexing and genotyping than is possible using LAMP or PCR assays. Fig.19 In an exemplary embodiment described in , twelve sensors were spotted with duplicates of six R-bead DENVPNA probes (PNA 1-6). These sensors include Fig.16 Two PNA probes for 100% dengue detection and one probe each for DENV 1 / 2 / 3 / 4 mutation identification. Fig.15Nine sensors were spotted with E and NS1 peptide sequences for highly specific DENV antibody detection: EP1, EP2, EP4, EP7, EP10, EP12, NS1-1, NS1-3, NS1-4. The bead quantification capability of each of these sensors can be used to establish the amount of E and NS1. Combined with the machine learning capabilities of CMOS chip electronics and processors, this can help improve assay sensitivity and selectivity, and help predict the potential onset of 'severe dengue' and severe and potentially fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Two PNA positive control sensors are included (if it is a blood sample, they are all spotted with 18s rRNA complementary probes, or if it is a saliva sample, they are spotted with RNase P complementary probes). Two 'PNA negative' negative control sensors are included, such as for single base mismatch confirmation test controls. Sensors spotted for IgM and IgG positive controls and aG negative antigen controls are also included. Two blank sensors for temperature and CMOS process variation correction and offset elimination are used.

[0101] This highly multiplexed and unique simultaneous detection capability of nucleic acids / antibodies in the assay is enabled by thirty co-located sensors and side-by-side fully synthetic peptide sequences and peptide-nucleic acid (PNA) probes, e.g. Fig. 20 As shown, each was synthesized via the same FMOC chemistry.

[0102] In certain embodiments, flow-through detection can be used to perform the multiple detection described above. For example, each light spot described above may not be a sensor. On the contrary, the tethering of the R beads to the substrate can be mediated by a carefully designed PNA joint or ligand sequence that does not interact with the RNA in the sample. These R beads are conjugated with a PNA probe or antigenic peptide sequence targeting RNA. The careful design of the joint PNA allows the R beads to be sequentially and selectively eluted from the substrate when the system is heated and the melting temperature of the joint PNA is reached. In this way, the R beads connected to the light spot mediated by the RNA target can be selectively and sequentially released and then measured on the downstream sensor. In addition, when the Tm of the relevant joint PNA is reached, the R beads connected to the light spot mediated by the antibody can be selectively released and measured on the downstream sensor. The sensor can be an embedded sensor that detects the R beads flowing through them. In certain embodiments, the sensor can be a PNA functionalized sensor combined with a specific R bead. Alternatively, the substrate can be the surface of a magnetic bead.

[0103] Testing of Bead Flow in Silicone Oil :

[0104] Fig.21 Depicts the CMOS silicon chip Figure 6 Another embodiment of a sensing capacitor of FIG. 1 is shown. In this embodiment, a sigma-delta capacitance-to-digital converter is formed within the CMOS chip, to the left of the sensing capacitor. Multiple diode temperature sensors integrated across the chip ensure that differences in sensor readings due to temperature variations across the chip can be measured and compensated. The sensors are formed in rows and columns offset to facilitate integration with a droplet dispenser for some assay automation applications or multi-channel assay approaches, for example where upstream nucleic acid and antibody assays can be in different channels.

[0105] Fig. 22 yes Fig.21 Photo of a chip with a transparent PD5 silicone oil film on some sensors of different diameters on the right half of the chip. The oil film is located in the fringe field transduction part of the IDE electrode capacitive sensor and thus increases the sensor capacitance due to the dielectric constant K of the oil being about 2.5 to 3, which is higher than air (K=1).

[0106] Fig.23 is a schematic diagram of a cylindrical portion of an oil film sensed over one of the circular sensors. Fig.24 Depicted Fig.23 The equivalent circuit shows the capacitance C of the insulating nitride passivation layer 氮化物 The oil capacitor C in series 硅酮 -Oil.

[0107] Fig.25 is a photograph of a PD5 oil film containing titanium dioxide (TiO2) beads on one of the circular sensors. These titanium dioxide analyte beads in the oil film add additional capacitance because the dielectric constant of these beads is about 30 to about 100, which is much higher than the oil dielectric constant (2.5 to 3). In fact, at lower frequencies (100 Hz, according to Wypych et al., DOI 10.1155 / 2014 / 124814), the K of TiO2 can be 10 times higher (up to K = 1000). Therefore, a further 10X assay amplification can be performed by slowing down the sigma-delta converter modulator frequency at the expense of a slower conversion time of up to 1 second.

[0108] Fig.26 yes Fig.25 Schematic diagram showing a cylindrical section of oil with beads on a circular sensor.

[0109] Fig. 27 yes Fig.25 and Fig.26 The equivalent circuit of the analyte sensor depicts the 氮化物 Series related C 硅酮-油 and C 珠粒 capacitance.

[0110] Fig.28 Sensor measurements of oil capacitance with and without beads are provided. At time zero, all sensors were zeroed with an air dielectric as the baseline. After 2 minutes, a film of PD5 oil (no beads) was applied to sensors S0 and S30 (as a negative control), and one minute later PD5 oil containing 1 μm TiO2 beads was applied to S6 and S25. As shown at the top of the graph, the beads increased the capacitance by 7.6 fF. Beads were visually counted on a microscope (see Figure 2). Fig.25 Photo) gives an estimate of about 6000 beads, indicating about 1.26 aF per bead. For a switched capacitor voltage of 1.6 V applied between the electrodes by the modulator of the sigma-delta converter, this is equivalent to about 12 electrons.

[0111] Referring to the S0 and S30 negative control sensors (0.96mm diameter), the addition of silicone oil at 2 minutes increased the capacitance by 132fF. For sensors S5 and S7, which are 0.54mm and 0.3mm in diameter, the capacitance increased by 45fF and 12.5fF, respectively. These measurements are summarized in Table 1 below, which shows that the silicone oil film adds approximately 183fF / mm2 compared to the air dielectric, regardless of sensor diameter:

[0112] Table 1

[0113]

[0114] These measurements of beads in oil illustrate various embodiments of the systems and methods of the present invention. They can be used to eliminate the need for liquid evaporation and are advantageous in allowing the sensor to be reused for multiple measurements. It can allow the beads to be moved by hydraulic flow control and injection, which is useful for non-magnetic beads and particles or magnetic particles to be detected. Fig.28 The graphs depicted in also show the change in capacitance as the oil film thickness varies and as the beads flow away from the sensor, demonstrating the dynamic real-time bead flow detection capability.

[0115] Electrical characterization of single beads and bead streams in oil :

[0116] Fig.29 Depicted are 0.4 μm beads in a 0.8 μm trench between electrodes.

[0117] Fig.30 COMSOL electrostatic simulations of the electrodes with 1 volt applied and of the bead (K=100) being transferred between electrodes in oil (K=2.5) are shown. The electric field gradient lines are shown in 0.1V steps.

[0118] Fig.31 COMSOL capacitance calculations of beads as they pass between electrodes are shown for bead dielectric constants of 2.5, 10, 30, 100, and 1000. The capacitance peaks at 4 attofarads (aF) when a 0.4 μm bead (K=100) in oil passes through an electrode gap of 0.8 μm. The capacitance of the 0.4 μm bead is higher than the capacitance of the larger 1 μm TiO2 bead of 1.2 aF (from Fig.28 ). This can further contribute to the overall signal amplification of this non-enzymatic detection / quantification assay. Increasing the electric field across the bead increases the dipole polarization of the molecules within the bead, thereby increasing its capacitance. The baseline capacitance of 399aF is quite low in this simulation, partly due to the short electrodes in the model without other wires or ground planes nearby, such as Fig.32 The boundary element model (BEM) used for this simulation is shown in FIG.

[0119] In practice, the electrodes may have wires a few millimeters in length that connect them to the capacitance-to-digital converter, and / or may be formed on a physical substrate (e.g., a 3D printed base or a CMOS silicon substrate). These substrates may behave electrically as a large ground plane. This Fig.33 , where a 0.4 μm bead passes through an electrode gap of 0.8 μm with a ground plane 2 μm below, which might be typical on a CMOS silicon substrate. For a K=100 bead in oil (K=2.5), the COMSOL bead capacitance is again 4 aF, as Fig.34 However, due to the increased parasitic capacitance to the nearby ground plane, Fig.34 The baseline capacitance is 4.274 femtofarads (fF) (i.e., Fig.31 10 times the baseline capacitance). Figure 1 and 4 In the embodiment shown in , longer wires (e.g., 10mm to 15mm) can further increase this baseline, for example, by 4.5pF to 6.1pF. It is noteworthy that even with these huge baseline changes from attofarads (aF) to picofarads (pF), a 4aF single bead capacitance is detectable. This is due to the wide dynamic range of the sigma-delta 24-bit capacitance-to-digital converter. This embodiment has a full-scale range of 8pF, a resolution of 0.5aF, and an accuracy of 4aF. Additional internal offset capacitors can be added to C 参考 (exist Figure 3In the example of FIG. 1 , the capacitance sensor can be fabricated in many different ways on a silicon substrate or CMOS chip by 3D printing with or without a ground plane, even in the presence of large parasitic effects.

[0120] Fig.35 A time domain plot of the converter output is shown for very low attofarad level measurements. Significant thermal and 1 / F noise is evident. A bead passing between electrodes may appear as a short 'blip' corresponding to a capacitance signal of approximately 3aF to 6aF (circled tip in waveform). Distinguishing these blips is not straightforward. Simple peak detection may miss ambiguous blips (in Fig.35 In some embodiments, moving average filtering can be used to help distinguish, but short spikes may be missed. Therefore, in some embodiments, multiple electrodes can be used in the bead channel to help by reading the same bead multiple times, such as Fig.36 As shown. A maximum likelihood estimation (MLE) machine learning algorithm can then be applied to the readout to improve bead counting accuracy. Partial response maximum likelihood (PRML) and Viterbi algorithms further improve bead discrimination and reduce error rates. These digital filtering improvements in signal-to-noise ratios, which can be embedded within the CMOS sensor chip itself, can further aid in overall assay amplification in the absence of enzymatic amplification.

[0121] Many amplification methods and steps have been described in this disclosure: PNA high capture efficiency, bead quality, CMOS10 6 The combined effects of electronic amplification, multi-bead tethering signal amplification, bead electric field enhancement in the trench, MLE, and PRML / Viterbi digital signal processing are sufficient to achieve 10 9 Up to 10 12 times amplification. This is roughly equivalent to 30 to 40 PCR cycles, respectively. Therefore, the fully synthetic assay described herein can be used to achieve an analytical sensitivity level of <100 copies / mL, but with a wide dynamic range of several levels, which is ideal for clinical RNA viral load monitoring. The assay of the present invention can further eliminate the requirement for enzymatic amplification, thereby also eliminating many inhibitory problems and complexities of PCR and LAMP assays. Due to being fully synthetic, the assay also eliminates the requirement for dry ice transportation and storage in a refrigerator or freezer. Relative to enzymatic assays where reagents can deteriorate faster and do not require special treatment, a long shelf life (possibly up to several years like electronic devices) is another possible benefit.

[0122] Through-hole bead flow assay

[0123] Fig.36 The beads are depicted flowing along channels or trenches on the substrate between multiple electrode pairs and then flowing down holes in the substrate, exiting at the rear. In some embodiments, this may be a DRIE etched through silicon via, as described in WO2004 / 109770.

[0124] Fig.37 An exemplary laser-formed through-hole in an ink electrode and a 3D-printed PLA substrate is shown. This is advantageous for in-line bead measurements, where beads can flow in tubes or channels.

[0125] Fig.38 Depicted is a narrow 0.8 μm electrode spacing between A and B ink-printed electrodes, formed by laser ablation with a 0.8 μm laser spot size. Beads flow in the channel and then 'drop down' through the holes between the electrodes (created by laser ablation of the 3D printed base). Smooth bead flow can be enhanced by injectable push-pull control of the oil or other liquid stream carrying the beads and / or by dielectrophoretic steering, where charged beads (e.g., -30 mV zeta potential) respond to positive or negative voltages applied from outside or along the flow channel (e.g., by Fig.38 Agglomeration of beads can be minimized by adjusting the zeta potential of the beads, for example to -30 mV via a carboxyl coating, which can cause the beads to repel each other, and / or by ultrasonically agitating the device to aid in monodisperse flow through the sensor.

[0126] In order to provide multiplexing capability in the flow detector system, multiple flow sensors can be placed in the same device. Corresponding to each variant to be tested, several discrete R bead groups of different sizes can be used. These R beads can be separated in space to allow each size group to be delivered to different through-hole sensors. The separation method can be carried out by inertial separation, dielectrophoretic separation or magnetophoretic separation. These beads can thus be distinguished, thereby allowing the corresponding target of each bead to be differentially detected in multiple determinations.

[0127] Recycling of beads

[0128] In certain embodiments, depending on the workflow, it may be advantageous to recirculate the beads back through the system. Fig.39 An exemplary bead recycling scheme is shown in . System 3901 has four main areas, sample capture 3903, assay construction 3905, elution 3907, and sensor detection 3909. Depending on the sensing method chosen, recycling T beads may have advantages because there is a continuous supply of T beads until the target RNA is depleted ( Fig.39). At 3903, the T beads capture the target RNA via the T probe. At 3905, the R probe and R beads are added in the next stage of assay construction, at which point the entire assay is magnetically transported through the sensor to the elution point 3907. The eluted R beads are then attached to the sensor 3909 via specific PNA-PNA binding. Over time, the number of R beads accumulates on a particular sensor until the source target RNA is no longer available. The final (total) signal is then captured. The T beads can be recycled 3911 after the elution step 3907 and returned to the initial capture step 3903, as Fig.39 The T beads may be cleaned before returning to the initial capture step 3903 to ensure that no dummy probes, R beads, or RNA are attached to the beads.

[0129] In the case where the CMOS detector is a single bead continuous flow sensor as described above, the assay set-up and elution follow the same steps with T beads recycled after elution, without sensor surface binding. In such embodiments, the R beads and T beads are transferred after elution, with the R beads flowing through the single bead CMOS detector and the T beads returned to the sample at the initial capture step until the target RNA is depleted. The signal is then read at each event to effectively count the number of beads that have passed through the CMOS detector.

[0130] Single molecule analysis :

[0131] As discussed throughout, various signal amplification techniques can be used alone or in combination to increase sensitivity, for example to allow single molecule detection. Single molecule detection has many applications, including early detection of infection and accurate quantification of viral load in, for example, the SARS-CoV-2 outbreak. Fig.40 An example of a single molecule analysis of, for example, SARS-CoV-2 is shown.

[0132] In early 2022, Lai et al. (DOI: 10.1101 / 2022.01.08.22268865) published a list of 48 markers or nucleotide mutations in the SARS-CoV-2 genome corresponding to the then-known variants: α, β, γ, δ, λ, μ, ε, ι, η, κ, and early Omicron variants. Assuming a 'brute-force' set of 48 probes targeting each of these mutation regions in the genome, Probe 1 is a capture probe designed to uniquely target all SARS-COV-2 variants, such as Fig.12As shown. Probe 1 can be attached to the assay's paramagnetic transport (T) beads, which flow continuously to capture and extract SARS-CoV-2 RNA from the sample. R beads can also flow sequentially into the tethered chamber in groups with different probes attached: first probe 2, then probe 3, then probe 4, and so on, up to probe 48. Variants in patient samples can then be identified by searching and narrowing the analysis, as Fig.13 and 14 In some embodiments, the number of probes required can be reduced by applying a 'truth table' logic reduction analysis to identify variants, such as Fig.41 As shown, where L452R, T478K, N501Y and H655Y mutations were mapped to identify minor variants.

[0133] Fig.41 An assay embodiment of a home breath test from a self-testing saliva sample is presented. In addition to the COVID-19 detection and variant identification of SARS-CoV-2 described above, additional probes are added to differentiate and identify influenza A, influenza B, RSV, and other viral pathogens, and peptide sequence probes for epitope antigens for detection of immunoglobulin E (IgE) antibodies are also added to identify allergic rhinitis ('hay fever'), which may be a cause of sneezing and congestion. The high single molecule assay sensitivity, lack of enzymatic amplification (which may be complicated when used at home), and multiplexed properties across target classes of the embodiments discussed above can all be combined to perform such a test.

[0134] Detection of beads flowing in other low dielectric constant liquids :

[0135] Other examples of low dielectric constant liquids are twelve fluoro-2-methylpentane-3-ketone (Novec 649) and fluorinated carbons, such as methoxy-nonafluorobutane (Novec 7100). These liquids are clear, colorless and low odor liquids that are used as advanced heat transfer fluids with desired environmental and electrical properties. Novec 649 is commonly used for electronic device cooling. It has a very low dielectric constant of 1.8 (at 1kHz), making it a favorable carrier medium for bead capacitive sensing. Additionally, it is dense and insoluble in water, and as a fluorinated substance, it will separate from aqueous and oil-based liquids. In certain embodiments, the beads released into the flow through the sensor can be monodispersed in the liquid to help determine sensitivity and single molecule detection. In certain embodiments, phase change can be used because the sample type is typically aqueous.

[0136] Fig.42A scheme for producing fluorinated R beads at the interface between an aqueous solvent and a fluorinated solvent according to certain embodiments is shown. The beads can be modified with ligands that allow transfer across the interface. The interface is shown in Fig.42 In the method, the beads are then modified into fluorinated R beads using, for example, heptafluorobutylamine (demonstrated with Dynabeads) and the beads are modified with fluoro-PNA (e.g., fluoro-PNA2 will form micelles and / or align at the aqueous / fluorinated fluid interface). This scheme illustrates how two-phase soluble PNAs (PNA and fluorinated end) can be used to label aqueous dissolved R beads, allowing the R beads to be subsequently transferred to fluorinated solvents. Similar chemicals can be used to produce lipophilic beads for transfer to oil. In some embodiments, the R beads can have amphiphilic properties and can be monodisperse in both aqueous solvents and organic or fluorinated solvents. Additionally, the use of amphiphilic detergents dissolved in one or both solvents can promote the transition of beads between solvent phases. Wei et al. (DOI: 10.1021 / ja039874m, incorporated herein by reference in its entirety) have demonstrated this method for transferring gold nanoparticles from an aqueous phase to an immiscible ionic liquid.

[0137] In certain embodiments, the transfer of beads to, for example, a final fluorinated solvent can be performed by transitioning between a variety of solvents (e.g., aqueous to aprotic / organic solvents, to fluoroester / ester / ketone solvents, to fluorinated or low dielectric solvents). Fig.43 The microfluidic method shown in Figure 1 performs this transfer. When the beads move across the solvent phase, jet technology can be used to introduce different solvents with overlapping mixing or micelle formation. The method can also facilitate the use of phase transfer molecules or amphiphilic detergents in each solvent in a separate solvent. The method is guided by the Hansen solubility parameters of the beads and the solvent to obtain sufficient solubility in each solvent to promote bead phase transfer. Similar methods can be used for microfluidic bead generation to transfer beads from organic solutions to aqueous solutions.

[0138] Another way to move the R beads from one solvent to another could be to utilize their high dielectric constant or high zeta potential to deflect the R beads from the flow of the "sample" or aqueous solution into a parallel flow of the Novec 649 or other fluid where the beads move laterally across the flow line until they are in the Novec 649 and deflected to the outlet of the sensor.

[0139] In some embodiments, magnetically sensitive R beads can be deployed in the device. As shown in the literature (Pamme and Manz, 2004, DOI10.1021 / ac049183o, incorporated herein by reference in its entirety), magnetophoretic separation can be used to move beads through a fluid. Magnetophoretic separation can be used to deflect R beads from an aqueous phase to a Novec 649 or oil-based phase or other phase.

[0140] When the beads are in the oil-based phase, the transfer of the beads from the aqueous phase to the oil-based phase allows a thin shell or droplet of aqueous solution to form around the beads. In some embodiments, flowing the aqueous coated beads through a capacitive sensor can provide an amplified signal that exceeds that of a standard bead due to the high dielectric constant of the aqueous shell on the beads, thereby further improving system sensitivity.

[0141] Tethered and Bead Flow Assay Combination :

[0142] exist Fig.44 In an embodiment, R beads eluted from an upstream assay can then be bound to additional beads to amplify the signal ('D beads', again simulating dendritic amplification). This can be achieved by using PNA-PNA binding between beads. This allows the R beads to be smaller and allows more R beads to be attached to a single molecule (e.g., RNA). Fig.45 An R-bead-D-bead complex is shown being measured across a flow channel, where two CMOS sensors are arranged on top of each other to partially or fully form the flow channel, and as the bead moves past each sensor, the capacitive signal is measured independently by the two chips. The measurement of one sensor is digitally compared to the measurement of the other sensor. The same arrangement can be used to measure individual beads.

[0143] R beads can be used to mediate the capture of R beads and D beads complexes on a substrate. The complex can have properties that mediate differentiation from uncomplexed beads. These properties can include capturing complexes on a substrate (e.g., mediated by R bead PNA), which can complete the determination that the substrate is the surface of the sensor. The properties of the complex can include PNA-PNA binding, dielectrophoresis, or electrophoresis. This property can attract the complex to the sensor. This property can cause the complex to deviate within a flow channel, where the complex passes through the sensor and uncomplexed particles flow through the outlet. This property can temporarily retain the complex or elements of the complex on the substrate, while the uncomplexed particles are washed and / or removed through the outlet. For example, in the case of using PNA-PNA bound to a substrate, after the uncomplexed particles are removed through the outlet, the temperature of the system can be increased to elute one or other members of the complex from the substrate, after which it flows through the sensor and can be measured as described elsewhere herein. In a flow-through embodiment, the capture and subsequent release of multiple beads can achieve the aforementioned tethered signal amplification.

[0144] In certain embodiments, the R beads can be reduced to a single PNA, and the binding of this PNA to another substrate can be used to mediate the capture of the D beads. Fig.46a As shown, the substrate can be a sensor chip (i.e., functionalized with PNA for PNA-PNA capture), or as Fig.46b As shown, the substrate can be additional magnetic beads. This can provide tethering and magnetic manipulation of the beads, with the advantage of having very strong PNA-PNA binding within each member of the tethered complex. Thus, when using a flow-through sensor, the advantages of signal amplification by tethering can still be obtained.

[0145] Fig.47 is an example of a much smaller R bead (1-100 nm) tethering a D bead to a surface.

[0146] Fig.48 Shown that the R nanoparticle can have other shapes, or a linker-type molecule that allows one or more probes to be connected, or a long PNA probe with two regions. Probe 2 can be a probe or ligand that is also functionalized on the R nanoparticle but does not interact with the natural RNA target. This can be achieved by careful probe design to avoid finding sequences in the natural RNA target and to avoid interacting with the PNA probe. The ligand can make it not interact with the normal base pairing on the target RNA, including but not limited to biotin-streptavidin, such as Fig.49 shown.

[0147] Fig.50 Shown are reduced size R nanoparticles (1-100 nm) tethering D beads to the surface of a magnetic particle for further manipulation prior to sensing.

[0148] Incorporated by Reference

[0149] Other documents, such as patents, patent applications, patent publications, journals, books, papers, web page content, have been referenced and cited throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0150] Equivalent

[0151] Various modifications to the invention and many other embodiments of the invention in addition to the embodiments shown and described herein will become apparent to those skilled in the art from the entire contents of this document (including references to the scientific literature and patent literature cited herein). The subject matter herein contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and its equivalents.

Claims

1. A method for nucleic acid detection, the method comprising using a capacitive sensor and detecting a target nucleic acid present in a sample at 100 copies / mL or less without enzymatic amplification.

2. The method of claim 1, which is operable to achieve at least 10 9 Second signal amplification.

3. The method of claim 1, operable to achieve a signal amplification equivalent to at least 30 PCR cycles in the detection of the target nucleic acid.

4. The method of claim 1, wherein the full-scale range of the nucleic acid detection performed by the capacitive sensor is at least about 8 pF. 5 . The method of claim 4 , further comprising converting a signal from the capacitive sensor using a capacitance-to-digital converter. 6 . The method of claim 5 , wherein the capacitance-to-digital converter is a sigma-delta 24-bit capacitance-to-digital converter.

7. The method of claim 4, wherein nucleic acid detection by a capacitive sensor comprises a resolution of at least about 0.5 aF and an accuracy of at least about 4 aF.

8. A method according to claim 4, wherein the full-scale range of the nucleic acid detection performed by the capacitive sensor is at least about 15 pF by including an internal offset capacitor in the reference input.

9. The method according to claim 1, further comprising: binding the target nucleic acid to reporter (R) beads; passing the R-bead through a sensor region including two capacitive electrodes on a substrate in communication with a signal processing circuit, wherein the two electrodes are spaced apart to form a gap, and wherein only a single bead moves between the gaps at a time; and The R beads are detected using the signal processing circuit as the R beads pass through the gap.

10. The method according to claim 9, further comprising: passing the R beads through a plurality of sensor regions; as well as The R beads are detected as they pass through the multiple sensor areas by applying one or more of a maximum likelihood estimation (MLE) machine learning algorithm, a partial response maximum likelihood (PRML) algorithm, or a Viterbi algorithm to signals received by the signal processing circuit from the multiple sensor areas.

11. The method of claim 1, wherein detecting the target nucleic acid comprises: binding the target nucleic acid to reporter (R) beads; as well as The R beads are detected using a capacitive sensor. 12 . The method of claim 11 , wherein detecting the R-bead using the capacitive sensor comprises detecting the R-bead flowing past the sensor in a fluid.

13. The method of claim 12, wherein the fluid comprises oil.

14. The method of claim 13, wherein the oil is silicone oil.

15. The method of claim 12, wherein the fluid comprises a fluorinated carbon.

16. The method of claim 15, wherein the fluid is selected from dodecafluoro-2-methylpentan-3-one and methoxy-nonafluorobutane.

17. The method of claim 14, further comprising fluorinating the R-beads.

18. The method of claim 13, further comprising forming a water layer around the R-beads flowing in the fluid to amplify a detection signal at the capacitive sensor.

19. The method of claim 12, further comprising binding the target nucleic acid to the R-beads using bead-bound peptide nucleic acid (PNA).

20. The method of claim 19, wherein the bead-bound PNA is a ligand linker PNA.

21. The method of claim 1, further comprising detecting a plurality of different targets including at least the target nucleic acid present in a sample using one or more capacitive sensors.

22. The method of claim 21, wherein the plurality of different targets comprises a plurality of different target nucleic acids.

23. The method of claim 22, wherein the plurality of different target nucleic acids are derived from different pathogens or different variants of a pathogen.

24. The method of claim 23, wherein the pathogen is SARS-CoV-2.

25. The method of claim 21, wherein the plurality of different targets comprises proteins.

26. The method of claim 25, wherein the protein is an antibody.

27. The method of claim 25, wherein the protein is an antigen.

28. The method of claim 26, wherein the target nucleic acid is derived from Dengue virus and the antibody is a dengue-specific antibody.

29. The method of claim 21, wherein detecting the plurality of different targets comprises: binding each of the plurality of targets to a different bead; as well as Each of the different beads is detected using the capacitive sensor.

30. The method of claim 29, further comprising distinguishing the different beads based on different capacitance detection signals from the capacitance sensor.

31. The method of claim 21, wherein detecting the plurality of different targets comprises: Each of the plurality of different targets is bound to a different probe on the substrate.

32. The method of claim 31 , wherein the substrate is operably associated with one or more capacitive sensors.

33. The method of claim 31, further comprising binding one or more reporter (R) beads to each of the different substrate-bound targets.

34. The method of claim 33, further comprising: selectively releasing the R-beads from the substrate-bound targets based on the different substrate-bound targets bound by the R-beads; allowing the released R-beads to flow in a fluid through the capacitive sensor; as well as The released R beads flowing through the sensor are detected.

35. The method of claim 29, wherein each of the different beads has a different size.

36. The method of claim 35, further comprising microfluidically directing each of the different beads to a different capacitive sensor based on a size of each bead.

37. The method of claim 36, wherein the directing step comprises one or more of an inertial method, a dielectrophoretic method, or a magnetophoretic method.

38. The method of claim 11, wherein detecting the target nucleic acid further comprises: combining the target nucleic acid with a probe on a substrate; as well as A plurality of reporter (R) beads are bound to the substrate-bound target nucleic acid.

39. The method of claim 38, wherein the plurality of R beads bind to different sequences in the substrate-bound target nucleic acid.

40. The method of claim 38, wherein the substrate is operably associated with the capacitive sensor.

41. The method of claim 39, further comprising releasing the plurality of R beads from the substrate-bound target nucleic acid; allowing the released R-beads to flow in a fluid through the capacitive sensor; and The released R beads flowing through the sensor are detected.

42. The method of claim 9, wherein the two electrodes form a groove through which the R-beads pass.

43. The method of claim 1, wherein detecting the target nucleic acid comprises: binding transport (T) beads to the target nucleic acid in the sample to form a T-bead complex; combining the T-bead complex with reporter (R) beads to form an R-bead complex; eluting the R beads and the T beads from the R bead complex; Detecting the R beads using a capacitive sensor; and The T-beads are returned to the sample to bind to another target nucleic acid.

44. A system for nucleic acid detection, the system comprising: A capacitive sensor, the capacitive sensor comprising: signal processing circuitry; and a sensor region comprising two capacitive electrodes on a substrate in communication with the signal processing circuit, wherein the two electrodes are spaced apart to form a gap, Wherein only a single bead moves between the gap at any time, and the electrodes detect the bead and / or any molecules attached to the bead.

45. The system of claim 44, wherein the system is reusable after being used to detect beads.

46. ​​The system of claim 44, wherein the capacitive sensor is approximately 2 mm x 2 mm.

47. The system of claim 44, wherein when beads or particles are contained in water, buffer, or oil, or fluorinated carbon or organic solvent and flow through the sensor area.

48. The system of claim 47, wherein the beads or particles detected are surrogates for microbial analytes from an upstream assay.

49. The system of claim 47, wherein the beads or particles can be magnetic or non-magnetic.

50. The system of claim 44, wherein the gap is 2 times the diameter of the beads.

51. The system of claim 44, wherein the substrate is 3D printed.

52. The system of claim 51, wherein the electrodes are formed by sputtering or ink deposition and patterning.

53. The system of claim 52, wherein the spacing between electrodes is about 0.8 μm to 20 μm.

54. The system of claim 44, wherein the substrate is a CMOS semiconductor chip.

55. The system of claim 54, wherein the electrodes are etched in a metal layer, wherein the spacing between electrodes is 40 nm to 5 μm.

56. The system of claim 44, further comprising one or more of ultrasonic agitation, dielectrophoretic bead steering, or adjustment of bead zeta potential to prevent bead agglomeration and / or oil ejection.

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