Method and device for determining bacterial concentration

Abstract

The present disclosure provides a method for determining a bacterial concentration in a sample, the method comprising the steps of: (a) introducing (i) a target droplet comprising the sample and (ii) a reference droplet comprising a control medium into an input region of a microfluidic device, the input region comprising a first input channel and a second input channel, wherein the target droplet is introduced into a carrier fluid in the first input channel and the reference droplet is introduced into the carrier fluid in the second input channel; (b) flowing the target and reference droplets from the input region into a synchronisation region to synchronise a pairing of the target and reference droplets; (c) flowing the target and reference droplets from the synchronisation region into a detection region to obtain a raw impedance signal for the pair of target and reference droplets; (d) calculating a calibrated impedance value by dividing a peak impedance value of the raw impedance signal corresponding to the target droplet with a peak impedance value of the raw impedance signal corresponding to the reference droplet; and (e) determining the bacterial concentration from the calibrated impedance value.

Description

DESCRIPTIONTITLE OF THE INVENTION: METHOD AND DEVICE FOR DETERMINING BACTERIAL CONCENTRATIONFIELD OF THE INVENTION

[0001] The invention generally relates to the field of microfluidics, and more particularly relates to a method and device for determining bacterial concentration.BACKGROUND OF THE INVENTION

[0002] The meticulous monitoring of bacterial growth kinetics, encompassing parameters such as bacterial growth rate, initial bacterial concentration, and doubling time, is essential across medical, industrial, and environmental domains. In clinical contexts, it consolidates timely diagnostics and antibiotic susceptibility assessments. Industrially, it is pivotal for optimizing fermentation processes and augmenting biotechnological yields. Environmentally, it facilitates effective wastewater treatment and bioremediation. Moreover, the analysis of growth kinetics is instrumental in constructing predictive models, thereby elucidating microbial behavior for enhanced process control. Such monitoring propels scientific understanding and advancements in public health, industrial efficacy, and environmental sustainability, underscoring its integral role in microbial research and applications.

[0003] The study of bacterial growth kinetics has progressed with the development of various monitoring techniques, each with distinct advantages and applications. Traditional methods, such as optical density measurements using spectrophotometry, remain widely utilized due to their simplicity and rapidity. However, they provide only bulk measurements, lack single-cell resolution, and have limited sensitivity to low bacterial concentrations. Advanced microscopic counting techniques allow for single-cell analyses but can be timeconsuming, low-throughput, and require sophisticated equipment. Plate counting remains a gold standard for assessing bacterial viability but is labor-intensive and entails extended incubation periods.

[0004] Recent advancements have ushered in real-time monitoring technologies. For instance, impedance biosensors have emerged as viable tools for real-time monitoring of bacterial growth by assessing the electrical impedance of the culture medium. Biochemical sensors have shown significant promise in providing rapid, real-time insights into bacterial growth kinetics by detecting physicochemical changes induced by bacterial activity andmonitoring changes in pH value, ATP level, consumption of O2 and production of CO2. However, these techniques primarily monitor changes of biochemical signals in bulk, leading to an inability to indicate accurate initial sample concentrations and exhibiting limited sensitivity in monitoring the growth of low concentrations of bacteria. Furthermore, surface modification of biochemical sensors in these methods is required, which may reduce their reliability and robustness. Microfluidic fluorescence-based flow cytometric assays have been proposed to address the aforementioned issues by offering the resolution of single-bacteria screening. However, given that fluorescence staining is laborious and the high complexity of laser-based screening systems, a fluorescence-free approach is desired. Advancing bacterial growth monitoring techniques amplifies the capacity for nuanced understanding and control of bacterial behavior across various fields, paving the way for enhanced microbial process management and infection control.

[0005] There is thus a need for a rapid, accurate and cost-effective method for determining a bacterial concentration in a sample that overcomes the drawbacks of the prior art. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides a method for determining a bacterial concentration in a sample, the method comprising the steps of: (a) introducing (i) a target droplet comprising the sample and (ii) a reference droplet comprising a control medium into an input region of a microfluidic device, the input region comprising a first input channel and a second input channel, wherein the target droplet is introduced into a carrier fluid in the first input channel and the reference droplet is introduced into the carrier fluid in the second input channel; (b) flowing the target and reference droplets from the input region into a synchronisation region to synchronise a pairing of the target and reference droplets; (c) flowing the target and reference droplets from the synchronisation region into a detection region to obtain a raw impedance signal for the pair of target and reference droplets; (d) calculating a calibrated impedance value by dividing a peak impedance value of the raw impedance signal corresponding to the target droplet with a peak impedance value of the raw impedance signal corresponding to the reference droplet; and (e) determining the bacterial concentration from the calibrated impedance value.

[0007] Advantageously, the claimed method allows for the impedance value of individual droplets to be obtained; and the counting of the number of droplets having an impedance value higher than the reference gate. Because bacteria are encapsulated in a single cell, the number of droplet higher than the reference gate would allow for the determination of the bacteria concentration. The SYNC (as will be described in detail below) can determine if the target droplet comprises any bacteria or not. If the droplet comprises bacteria, then the target droplet should have a higher impedance value than the reference droplet. The average impedance value determines the bacterial growth kinetics, and the SYNC count determines the bacterial concentration.

[0008] In one embodiment, the sample comprises bacteria suspended in a culture medium.

[0009] In one embodiment, the control medium comprises a culture medium.

[0010] In one embodiment, the culture medium comprises peptone, yeast extract, trimethylamine N-oxide (TMAO) and glucose.

[0011] In one embodiment, the peptone is present at a concentration of between 0.6 and 5.4 g / L of the culture medium.

[0012] In one embodiment, the yeast extract is present at a concentration of between 0.3 and 2.7 g / L of the culture medium.

[0013] In one embodiment, the TMAO is present at a concentration of between 0.833 and 5 g / L of the culture medium.

[0014] In one embodiment, the glucose is present at a concentration of between 75 and 150 g / L of the culture medium.

[0015] In one embodiment, step (a) comprises forming the target and reference droplets directly into the carrier fluid in the input region.

[0016] In one embodiment, step (a) comprises pre-forming the target and reference droplets and subsequently introducing the pre-formed target and reference droplets into the carrier fluid in the input region.

[0017] In one embodiment, the target and reference droplets are formed via an encapsulation method selected from the group consisting of microfluidic emulsion generation, vortexing and pipetting.

[0018] In one embodiment, the pre-formed target and reference droplets are incubated for a predetermined duration before being introduced into the carrier fluid in the input region.

[0019] In one embodiment, the predetermined duration ranges from 1 to 5 hours.

[0020] In one embodiment, step (e) comprises counting a number of the calibrated impedance values falling within a predetermined impedance range, wherein a higher number of calibrated impedance values falling within the predetermined impedance range is indicativeof a higher bacterial concentration in the sample. This may be referred to as the “SYNC count” in this application.

[0021] In one embodiment, step (e) comprises averaging the calibrated impedance values falling with a predetermined impedance range, wherein a higher average value of the calibrated impedance values falling within the predetermined range is indicative of a higher bacterial concentration in the sample.

[0022] In one embodiment, the microfluidic device is the microfluidic device as described herein.

[0023] In another aspect, the present disclosure provides a microfluidic device comprising: an input region comprising a first input channel and a second input channel, the first input channel being configured to receive a target droplet and the second input channel being configured to receive a reference droplet; a synchronisation region comprising a first synchronising channel and a second synchronising channel, the first synchronising channel being in fluid communication with the first input channel and configured to receive the target droplet from the first input channel, the second synchronising channel being in fluid communication with the second input channel and configured to receive the reference droplet from the second input channelwherein the synchronisation region is configured to synchronise the pairing of the target and reference droplets; a detection region comprising: (i) a first detection channel and a second detection channel, the first detection channel being in fluid communication with the first synchronising channel and configured to receive the target droplet from the first synchronising channel, the second detection channel being in fluid communication with the second synchronising channel and configured to receive the reference droplet from the second synchronising channel; and (ii) an electrode arrangement configured to measure an impedance of the target and reference droplets.

[0024] In one embodiment, the microfluidic device as described herein further comprises a means for introducing the target droplet and reference droplet into the input region.

[0025] In one embodiment, the first and second synchronising channels are interconnected in a rail-like arrangement in which the first and second synchronising channels have substantially perpendicular interconnecting passages therebetween to allow fluid communication between the first and second synchronising channels, the interconnecting passages being configured to allow a carrier fluid to pass between the first and second synchronising channels, wherein the rail-like arrangement is configured to synchronise the pairing of the target and reference droplets.

[0026] In one embodiment, the first detection channel and the second detection channel are substantially parallel to each other.

[0027] In one embodiment, the electrode arrangement comprises a central electrode, a first sensing electrode and a second sensing electrode, wherein the central electrode, the first sensing electrode and the second sensing electrode are substantially perpendicular to the first and second detection channels, wherein the first and second sensing electrodes are disposed on opposite sides of the central electrode such that the first sensing electrode, the central electrode and the second sensing electrode are substantially parallel to one another, wherein the first sensing electrode and the central electrode are configured for measuring an impedance of the plurality of reference droplets and wherein the central electrode and the second sensing electrode are configured for measuring an impedance of the plurality of target droplets.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0029] Fig. 1 shows the SYNC technology for sensitive single-bacterial growth monitoring, a A schematic of the SYNC mechanism involving droplet emulsion and electrochemical modifications for superior sensitivity compared to traditional methods: droplet 10 is a reference droplet, and 20 is a target droplet, b The composition of a specially engineered bacterial culture medium that enhances electrical conductivity through bacterial metabolic activity, c The SYNC chip design featuring self-synchronization and electrical detection regions for accurate droplet characterization and signal processing, d Microscopic images illustrate the chip’s critical regions, e The process of electrical signal analysis is detailed in “Operation of SYNC” under the Material and Methods section, showing droplets with bacteria are distinguished from empty ones through signal processing. All scale bars in the figures represent 200 pm unless specified.

[0030] Fig. 2 shows the TMAO amplifier's impact on medium conductivity and bacterial growth, a Adding peptone and yeast extract to DI water in a 2:1 ratio (0.3 to 2.7 g / l) linearly increases medium conductivity (R2= 0.99). b Chemical reaction of TMAO. E.coli, a common type of facultative anaerobic bacteria, reduces TMAO to TMA (top). The hydrolysis of TMA generates hydroxide irons, leading to an increase in the conductivity of the medium (bottom), c A 20-hour incubation shows increased medium conductivity with TMAO up to 50 pl; beyond this, the increase diminishes. The conductivity modification in 20-hour incubation by varying TMAO and conductive nutrition (10 pl: 0.833g / L to 60 pl: 5g / L). d In a 5-hour study, 80 plpeptone (and 40 pl yeast extract) matches the MH medium's bacterial growth rate (OD600 = 0.1), while 20 pl peptone (and 10 pl yeast extract) outpaces PBS, affecting conductivity from 253 to 760 pS / cm and 805 to 1857 pS / cm, respectively, e Optimization of TMAO-amplified medium in its growth factor and conductivity. With the increase of the concentration from conductive nutrition, suspending bacteria grow faster (left), with also the increase of conductivity changes (middle). However, the conductivity change rate (final conductivity divided by the initial conductivity) decreases with more conductive nutrition (right). Thus, the optimized concentration of conductive nutrition is adding from 20 to 60 pl of peptone stock solution, with 10 to 30 pl of yeast extract stock solution, respectively, f-g A 12-hour incubation tracking growth via conductivity and optical density (ODeoo = 0.02 to 0.2) shows conductivity correlating with bacterial growth, confirmed by optical density, with high linearity (R2= 0.97 to 0.91 ) across varying bacterial concentrations. Data are presented as mean + s.d. (standard deviation). Fig. 2f includes two different conditions of initial bacterial concentration (low: left; high: right). This shows that the culture medium of this invention can determine the bacterial concentration based on conductivity in a good linearity with the gold standard method: OD600.

[0031] Fig. 3 shows the functionality of the SYNC chip, a The self-synchronization of droplets, despite their natural tendency to aggregate, is achieved through a rail-based structure that manages their flow and phase, ensuring synchronization with the paring efficiency of 95.8%. b Electrical simulations indicate that droplet conductivity, measured via AC voltage, significantly influences impedance changes, with higher conductivity leading to larger peaks, c This allows for the distinct identification and unbiased comparison of droplet conductivity data, with solutions of 60 and 70 pS / cm being effectively differentiated without the need for repeated sample loading (5756 droplets for the statistical analysis), d The SYNC chip's conductivity detection spans an extensive range from 23 to 1.6k pS / cm, exhibiting high linearity (R2= 0.99) with bulk conductivity and maintaining this linearity (R2= 0.97) within the key range for bacterial growth detection, with a resolution of approximately 100 pS / cm. All scale bars in the figures represent 200 pm unless specified.

[0032] Fig. 4 shows a detailed multiparametric analysis of the SYNC platform for real-time bacterial growth monitoring (E. coli). a Unlike traditional methods like optical density and plate counting, which lack sensitivity for low bacterial concentrations and involve laborious procedures, SYNC employs electrochemical measurements in bacteria-encapsulated droplets for immediate and highly sensitive growth detection, b In high-concentration samples (OD6oo = 0.2), SYNC differentiates between growing bacteria and empty droplets over time, with significant growth visible in impedance scatter plots after 3 and 5 hours of incubation (7257, 8921 , 8385 droplets for the statistical analysis for 0, 3, 5 hours respectively), c Analyzinggrowth rates using SYNC impedance and SYNC count reveals that SYNC count, with a slope similar to OD600 (k = 0.23 vs. k = 0.22), is a more reliable metric for growth than SYNC impedance, which has a lower slope (k = 0.15). d This method shows a strong linear correlation (R2= 0.88) between bacterial growth (E. coli) monitored by optical density (ODeoo = 0.37 to 1 .56) and SYNC count, underscoring SYNC'S effectiveness for precise bacterial growth analysis. Data are presented as mean + s.d. (standard deviation). All scale bars in the figures represent 50 pm unless specified. This invention provides a method for measuring the change of droplet impedance, which in turn measures or determines bacterial concentration.

[0033] Fig. 5 shows the capacity of SYNC to profile low-concentration bacterial (E. coli) suspensions, a-b The volume ratio is defined as the total volume of suspended bacteria to the suspension medium volume (1 ml), focusing on encapsulation probabilities with droplet volume at 380 pl, showing lambda values for concentrations at 107, 106, and 104bacteria / ml are 6.4, 0.64, and 0.0064, respectively, indicating a high rate of single bacteria encapsulation at concentrations <106bacteria / ml. At 104bacteria / ml, the single bacteria encapsulation rate drops to less than 0.6%. c In tests with 5 x 104bacteria / ml, theoretical and experimental singlet encapsulation probabilities are about 3% and 2.3%, respectively. This allows for observable population growth within a designated impedance target gate (7006, 6514, 6556 droplets for the statistical analysis in 0, 3, 5 hours respectively), d The limit of detection (LOD) for bacterial growth is refined to SYNC count, revealing SYNC'S 10-fold greater detection sensitivity compared to OD600, achieving statistical significance in detecting bacterial growth twice as fast as traditional methods, e SYNC demonstrates higher sensitivity in detecting the growth of E. coli in 2 hours, 50% faster than OD600. f A 4-hour incubation allows SYNC to accurately calculate initial bacterial concentrations comparable to 24-hour plate counting results. Data are presented as mean ± s.d. (standard deviation). All scale bars in the figures represent 50 pm unless specified.DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure provides a versatile system that leverages the electrochemical changes in culture medium from bacterial respiration and the advances of droplet microfluidics for bacterial growth kinetic monitoring. The present inventors engineered the bacterial culture medium to balance its conductivity and the growth nutrition of bacteria to amplify the detection sensitivity for bacterial growth. This medium also confers a label-free characteristic to the assessments undertaken to characterize bacterial growth. Additionally, a novel microfluidic device named SYNC (for 'self-synchronized droplet-amplified electricalscreening cytometry') has been integrated with this technology. The SYNC system in accordance with the present embodiments employs a dual-microfluidic channel structure for parallelly screening the target droplets that encapsulate bacteria and a blank reference droplet carrying the specially designed medium for enhancing the detection signal-to-noise ratio by calibrating individual droplets encapsulating bacteria with reference droplets. A rail-based droplet self-synchronization channel is utilized in the SYNC to support parallel screening due to its robustness and ease of fabrication. The SYNC system is robust and sensitive. By engineering the composition that supports bacterial growth, including the nitrogen source and carbon source, as well as introducing TMAO (trimethylamine N-oxide) as the electrochemical amplifier, the optimized bacterial culture medium has demonstrated a high linearity between bacterial growth and the conductivity of the bacterial culture medium, while retaining the growth rate compared to the MH (Mueller-Hinton) culture medium. By integrating droplet encapsulation and culturing, SYNC has shown a high linearity between the count of bacteria- encapsulated droplets in a preset target gate and bacterial growth verified by optical density. SYNC has also been utilized to monitor bacterial growth kinetics at a low concentration (5 x io4bacteria / ml), which is undetectable by optical density and can only be verified by plate counting. The system achieved sensitive quantification in growth rate monitoring (5-fold improvement compared with optical density), resulting in faster determination of bacterial doubling time and growth rate (50% faster). Additionally, SYNC demonstrated a unique feature never shown in any previous real-time monitoring approaches: providing the exact initial bacterial count in the original sample in 4 hours, as accurate as plate counting, which requires 24 to 48 hours for results.

[0035] The introduction of the SYNC system in accordance with the present embodiments provides an alternative approach to bacterial growth kinetics monitoring, merging droplet microfluidics with electrochemical amplification. In comparison to traditional methods, this integration seeks to address limitations in both speed and accuracy. Standard techniques, such as plate counting, although recognized for precision, necessitate extended incubation periods of up to 24 hours. Optical density methods offer quicker results, but their limited detection threshold can lead to inaccuracies, especially in low-concentration bacterial samples. The performance of SYNC has shown consistency across varying sample concentrations. Specifically, its evaluation of bacterial samples revealed growth rates comparable to those deduced using optical density. Advantageously, SYNC'S performance in the domain of low- concentration bacterial samples is significant. Traditional optical methods report detection thresholds of bacteria growth in approximately 106bacteria / ml. In contrast, SYNC sensitizes this range to 105bacteria / ml, with a 10-fold enhancement. Further advantageously, SYNC hasbeen shown not only to speed up 50% of bacterial growth detection in time for different kinds of bacteria, which could have implications for time-sensitive applications but also to deliver the exact initial bacterial concentrations, down to 104bacteria / ml, which has never been demonstrated in other electrochemical-based real-time monitoring platforms. It is worth noting that SYNC could extend the test time for slow growing bacteria, because of the nature of measurement in bacterial growth kinetics. However, it has demonstrated a higher sensitivity (50%) in bacterial growth detection compared to conventional optical density approaches.

[0036] In one aspect, the present disclosure provides a method for determining a bacterial concentration in a sample, the method comprising the steps of: (a) introducing (i) a target droplet comprising the sample and (ii) a reference droplet comprising a control medium into an input region of a microfluidic device, the input region comprising a first input channel and a second input channel, wherein the target droplet is introduced into a carrier fluid in the first input channel and the reference droplet is introduced into the carrier fluid in the second input channel; (b) flowing the target and reference droplets from the input region into a synchronisation region to synchronise a pairing of the target and reference droplets; (c) flowing the target and reference droplets from the synchronisation region into a detection region to obtain a raw impedance signal for the pair of target and reference droplets; (d) calculating a calibrated impedance value by dividing a peak impedance value of the raw impedance signal corresponding to the target droplet with a peak impedance value of the raw impedance signal corresponding to the reference droplet; and (e) determining the bacterial concentration from the calibrated impedance value.

[0037] In one embodiment, the sample comprises bacteria suspended in a culture medium.

[0038] In one embodiment, the control medium comprises a culture medium.

[0039] In one embodiment, the culture medium comprises peptone, yeast extract, trimethylamine N-oxide (TMAO) and glucose.

[0040] In one embodiment, the peptone is present at a concentration of between 0.6 and 5.4 g / L of the culture medium.

[0041] In one embodiment, the yeast extract is present at a concentration of between 0.3 and 2.7 g / L of the culture medium.

[0042] In one embodiment, the TMAO is present at a concentration of between 0.833 and 5 g / L of the culture medium.

[0043] In one embodiment, the glucose is present at a concentration of between 75 and 150 g / L of the culture medium.

[0044] In one embodiment, step (a) comprises forming the target and reference droplets directly into the carrier fluid in the input region.

[0045] In one embodiment, step (a) comprises pre-forming the target and reference droplets and subsequently introducing the pre-formed target and reference droplets into the carrier fluid in the input region.

[0046] In one embodiment, the target and reference droplets are formed via an encapsulation method selected from the group consisting of microfluidic emulsion generation, vortexing and pipetting.

[0047] In one embodiment, the pre-formed target and reference droplets are incubated for a predetermined duration before being introduced into the carrier fluid in the input region.

[0048] In one embodiment, the predetermined duration ranges from 1 to 5 hours.

[0049] In one embodiment, step (e) comprises counting a number of the calibrated impedance values falling within a predetermined impedance range, wherein a higher number of calibrated impedance values falling within the predetermined impedance range is indicative of a higher bacterial concentration in the sample.

[0050] In one embodiment, step (e) comprises averaging the calibrated impedance values falling with a predetermined impedance range, wherein a higher average value of the calibrated impedance values falling within the predetermined range is indicative of a higher bacterial concentration in the sample.

[0051] In one embodiment, the microfluidic device is the microfluidic device as described herein.

[0052] In another aspect, the present disclosure provides a microfluidic device comprising: an input region comprising a first input channel and a second input channel, the first input channel being configured to receive a target droplet and the second input channel being configured to receive a reference droplet; a synchronisation region comprising a first synchronising channel and a second synchronising channel, the first synchronising channel being in fluid communication with the first input channel and configured to receive the target droplet from the first input channel, the second synchronising channel being in fluid communication with the second input channel and configured to receive the reference droplet from the second input channelwherein the synchronisation region is configured to synchronise the pairing of the target and reference droplets; a detection region comprising: (i) a first detection channel and a second detection channel, the first detection channel being in fluid communication with the first synchronising channel and configured to receive the target droplet from the first synchronising channel, the second detection channel being in fluid communication with the second synchronising channel and configured to receive the reference droplet from the second synchronising channel; and (ii) an electrode arrangement configured to measure an impedance of the target and reference droplets.

[0053] In one embodiment, the microfluidic device as described herein further comprises a means for introducing the target droplet and reference droplet into the input region.

[0054] In one embodiment, the first and second synchronising channels are interconnected in a rail-like arrangement in which the first and second synchronising channels have substantially perpendicular interconnecting passages therebetween to allow fluid communication between the first and second synchronising channels, the interconnecting passages being configured to allow a carrier fluid to pass between the first and second synchronising channels, wherein the rail-like arrangement is configured to synchronise the pairing of the target and reference droplets. The first and second synchronising channels may be substantially parallel to each other. Alternatively, other suitable dual-channel active synchronization may be carried out, e.g. using surface acoustic wave induced droplet generation etc.

[0055] In one embodiment, the first detection channel and the second detection channel are substantially parallel to each other.

[0056] In one embodiment, the electrode arrangement comprises a central electrode, a first sensing electrode and a second sensing electrode, wherein the central electrode, the first sensing electrode and the second sensing electrode are substantially perpendicular to the first and second detection channels, wherein the first and second sensing electrodes are disposed on opposite sides of the central electrode such that the first sensing electrode, the central electrode and the second sensing electrode are substantially parallel to one another, wherein the first sensing electrode and the central electrode are configured for measuring an impedance of the plurality of reference droplets and wherein the central electrode and the second sensing electrode are configured for measuring an impedance of the plurality of target droplets.

[0057] Examples of carrier fluid include hexene, PBS or any low or non-conductive fluid.

[0058] The term “pairing” in the context of a pair of target and reference droplets is meant to refer to pairing one target droplet and one reference droplet such that the one target droplet and one reference droplet passes through the detection electrodes synchronously. By “synchronously”, it may mean that the target and reference droplets are within 1 ms of each other.

[0059] The synchronisation region may have passive or active synchronisation configurations. The rail-like arrangement, where carrier fluid can pass through the interconnecting passages but droplets cannot, is an example of passive synchronisation. In this rail-like arrangement, hydrodynamic force is used to push or pull individual droplets so that the target and reference droplets in each pair of droplets are synchronised.

[0060] It would generally be appreciated that the electrodes in the detection region do not need to be perpendicular to the detection channels in order for the invention to work. The electrodes also do not need to be in the shape of a bar in order for the invention to work. The electrodes may be below the detection channels.

[0061] The terms “central electrode” and “power electrode” are used interchangeably.

[0062] As used herein, the terms “target gate” and “predetermined impedance range” are used interchangeably. The target gate refers to the measurement of impedance value at 0 hour incubation. The target gate may also be defined as the impedance value of an empty droplet. As shown in this disclosure, the detection is based on the change in impedance value. The target gate is used for calibration. Therefore, it should be appreciated that the target gate is not essential if the test were to be calibrated each time before use.

[0063] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0064] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0065] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0066] MATERIAL AND METHODS

[0067] The composition of engineered bacterial culture medium

[0068] The engineered bacterial growth medium was formulated utilizing prepared stock solutions. The peptone stock solution was prepared by dissolving peptone powder (82450- 100G, Sigma) in deionized (DI) water to achieve a concentration of 100 g / l. Similarly, the yeast extract stock solution was prepared by dissolving yeast extract powder (Y1625-1 KG, Sigma) in DI water to reach a concentration of 250 g / l. It should be noted that both peptone and yeast extract serve as conductive nutrients, and were combined in a 2:1 volume ratio in this disclosure, unless otherwise specified. In the experiments, the volume of the conductive nutrients refers to the volume of the peptone stock solution. The TMAO stock solution was prepared by dissolving TMAO powder (T0514-100G, Sigma) in DI water to achieve a concentration of 250 g / l. The glucose stock solution was prepared by dissolving glucose powder (G5767-500G, Sigma) in DI water to achieve a concentration of 600 g / l. The engineered bacterial culture medium was then formulated by combining the requisite volumes of peptone and yeast extract, with 60 pl of TMAO stock solutions and 30 pl of the glucose stock solution in 3 ml of DI water.

[0069] Microfabrication and system setup

[0070] Microfluidic channels were fabricated using photolithography and soft-lithography on a 4" silicon wafer coated with SU-8 2015, followed by baking, UV exposure, development, and hydrophobic treatment with trichloro(1 H,1 H,2H,2H-perfluorooctyl) silane. PDMS was prepared, degassed, and molded on the master, then cured and processed to form the device structure. Electrodes were patterned on glass wafers using a lift-off process, depositing Cr / Au layers, followed by oxygen plasma treatment for PDMS-glass bonding. Precise alignment and bonding were achieved using an x-y-z positioner, with a final baking step to strengthen the bond. The detailed fabrication process and system setup is described in the paragraph below.

[0071] Microfluidic device fabrication and system setup

[0072] The creation of microfluidic channels adhered to the established norms of photolithography and soft-lithography techniques. Initially, a 4" silicon wafer was spin-coated with SU-8 2015 (MicroChem) at 2000 rpm, resulting in a photoresist film with a thickness of about 20 pm. Following the coating, a soft-bake procedure was executed at 65 °C for 1 minute and at 95 °C for an additional 3 minutes. UV exposure was then administered at a dose of 155 mJ / cmA2, after which a post-bake process at 65 °C for 1 minute and 95 °C for 3 minutes was carried out. The wafer was developed in SU-8 developer solution at room temperature. The resulting master mold underwent a vapor deposition treatment with trichloro(1 H,1 H,2H,2H- perfluorooctyl) silane (Sigma-Aldrich) to attain hydrophobicity. PDMS (Sylgard 184 silicone elastomer kit, Dow Corning) was utilized for the rapid prototyping process, with a mixture of base and curing agent (10:1 ratio) prepared, degassed, and poured onto the master mold.Following a curing process at 60 °C for a minimum of 3 hours, the solidified PDMS was removed from the mold segmented, and inlets and outlets were created using a biopsy punch (1 .5 mm diameter).

[0073] Electrode patterning was carried out on 4" glass wafers through a lift-off process, resulting in electrodes with a width of 20 pm and a spacing of 30 pm between adjacent electrodes within a single sensing pair. The Cr / Au electrodes (10 nm / 100 nm) were deposited on a 4" glass substrate. The surfaces of the PDMS and glass substrate were treated with oxygen plasma to enable permanent bonding. Utilizing an x-y-z positioner, a meticulous alignment of PDMS channels and electrodes was performed, followed by bonding. The bonded assembly was then subjected to a baking process at 120 °C for 1 hour to enhance bonding strength.

[0074] The SYNC system was initialized by priming with fluorinated oil (FluoSurf, Dolomite). Following this, target droplets and reference droplets were introduced into their respective channels. The flow rate of the spacing oil phase was meticulously adjusted to match that of the sample inlet. Upon setup, droplets were directed to the electrical screening region for analysis by the impedance analyzer. The impedance analyzer in use, HF2LI from Zurich Instruments, comes integrated with a lock-in amplifier and a microprocessor and is engineered to generate a 2V AC voltage at a frequency of 5 MHz, which is applied across the pairs of impedance sensing electrodes. In various embodiments, the frequency may be in the range of between 1 MHz to 40MHz. The microprocessor is tasked with digitizing the electrical current emanating from the differential current amplifier connected to the grounding of the sensing electrodes, executing this function at a sampling rate of 28.8 kHz. Following the acquisition phase, the electrical signals were relayed to a computing environment where they underwent an analytical process facilitated by MATLAB.

[0075] Operation of SYNC

[0076] SYNC leverages droplet emulsion (Fig. 1a, bottom left) and electrochemical modification from the incubation of single-bacterial droplets (Fig. 1 a, right) and demonstrates higher sensitivity in monitoring bacterial growth at low sample concentration in the early growing stages than conventional bulk optical density measurement (Fig. 1 a, top left). Fig. 1b illustrates the compositions of engineered bacterial culture medium. The medium can not only provide nitrogen and carbon sources as bacterial growth factors but also utilize TMAO as an electrochemical transducer and amplifier, converting bacterial metabolic activity to the growth of electrical conductivity of the medium. The SYNC chip schematics. The SYNC chip consists of two parts: the self-synchronization region (Fig. 1c) and the electrical detection region (Fig. 1c). A pair of droplets injected in the same frequency, but a different phase can besynchronized at the same position in the self-synchronization region. When the pair of synchronized droplets pass through the detection region, the reference droplet (Fig. 1 c, empty) is characterized first, and then the target droplet (encapsulating bacteria illustrated as rods in Fig. 1 c) is detected. Then, the electrical signal can be differentially amplified by the transimpedance differential amplifier (TA) and quantized by an impedance analyzer. Fig. 1d shows the microscopic images of the self-synchronized region and the parallel detection region. Note that the rail connectors and the detection region with electrodes have a channel height of 20 pm, while others are 40 pm.

[0077] The raw impedance signal indicates the real-time impedance signals quantized in time. It consists of the positive target droplet signals (Fig. 1e) and reference droplet signals (Fig. 1e).After extracting the peaks of the raw electrical signals, there are two clusters from the target droplets, representing one is the bacteria-encapsulated droplets and the other overlapping with the reference droplets are empty droplets. The total impedance data acquisition time is 5 minutes to ensure sufficient data points for statistical analysis. Then, the processed impedance data are unbiased by dividing the data from the target droplets to the corresponding reference droplets in individual pairs of screened droplets. The impedance data of empty droplets are unbiased at 1 (Fig. 1 e, dotted line).

[0078] Bacteria culture and preparation

[0079] Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) was prepared in accordance with the guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI). An aliquot of the bacterial culture was diluted in Cation Adjusted Mueller Hinton Broth (MHB) to achieve a target concentration of 106bacteria / mL in each tube. The concentration of the bacterial suspensions was ascertained by determining the extinction coefficients from the optical density readings, utilizing a spectrophotometer (NanoDrop One Spectrophotometers, Thermo Scientific). Subsequently, the E. coli suspension was subjected to three resuspension cycles in the engineered culture medium, each followed by centrifugation at 2000 ref for 10 minutes. Then, the resuspended bacterial suspension (20 pl) was emulsified to microdroplets with a diameter of 40 pm (about 6 x 105droplets were generated). After incubating for the required period of time, the microdroplets were injected into SYNC with the screening throughput over 1000 droplets / minute. The impedance data acquisition time for each measurement took 5 minutes for obtaining sufficient analytical data.

[0080] Numerical simulation

[0081] Electrical simulations help to understand the electrical characterization of individual droplets. The simulation was conducted utilizing the Electric Current (ec) module in COMSOL 5.0, employing 2D finite element modeling. The electrical parameters, along with the modeldimensions, are summarized in Table 1 . The fluidic channel, defined by dimensions of 180 pm in length and 20 pm in height, incorporates two rectangular electrodes, each with a thickness of 100 nm. The remaining boundaries of the fluidic region are characterized by electrical isolation.

[0082] Table 1 : Simulation parameters. Au is the material of the electrodes, which has an electrical conductivity of 45.6 x 106S / m.Table 1.

[0083] Statistical analysis

[0084] Data was demonstrated as mean with error bars representing standard deviations. The statistical difference is evaluated by the two-sample t-test in MATLAB, where n.s. is not significant, * p < 0.05, ** p < 0.01 , p < 0.001 . Over 1000 droplets were analyzed in statistical analysis, otherwise specified.

[0085] EXAMPLES

[0086] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0087] Example 1 : The SYNC technology

[0088] Optical density is a common metric employed for monitoring bacterial growth across various microbial-related applications. However, it was previously demonstrated that optical density falls short in measuring bacterial concentrations lower than 105bacteria / ml. Consequently, assessing growth kinetics in samples with low bacterial concentrations may yield inaccurate results, misrepresenting crucial parameters such as growth rates and doubling time, which are pivotal for discerning bacterial growth kinetics. To address this limitation, the present disclosure introduces the SYNC system, which leverages droplet emulsion technology to encapsulate single bacteria, thereby confining them in picoliterdroplets to maximize the volume ratio between bacteria and suspension medium (Fig. 1a). It would be appreciated that the target droplets could have zero, one or more bacteria. It would also be generally appreciated that the invention would work with the droplets having picolitre volumes as well as with larger than picolitre volumes, such as microlitre volumes. Due to the oil barriers surrounding the droplets, conventional biochemical sensors employing surface modifications, such as self-assembly monolayers for immuno-immobilization, become dysfunctional. To probe the bacterial growth kinetics within droplets utilizing the electrical sensors in SYNC, the present inventors devised a novel bacterial culture medium. This medium incorporates an amplifier, TMAO, to transduce bacterial metabolic activity into discernible changes in the electrical properties of individual droplets alongside proactive growth factors including peptone and glucose, serving as nitrogen and carbon sources, respectively, to support normal bacterial physiological activity (Fig. 1 b). Bacterial respiration reduces TMAO, generating hydroxide ions which elevate the conductivity of individual droplets, a process termed as phosphorylated amplification.

[0089] Reference particles are traditionally employed in impedance cytometry to calibrate raw impedance signals since impedance readings can fluctuate due to device-to-device variations and electronic wire connections. This necessitated repeated sample loading, posing a challenge in impedance cytometric assays over extended periods. To surmount this hurdle, the SYNC chip was designed with a dual-channel architecture, termed the parallel detection region, enabling simultaneous screening of target and reference droplets, and a rail-based structure for droplet synchronization dubbed the self-synchronization region. Upon injecting a pair of droplets into the microfluidic channel, they are automatically synchronized within the rail-based structure, thereafter being screened by three sensing electrodes situated beneath the dual-channel detection region (Fig. 1c). The reference droplets are initially screened in the bottom channel as they reach the sensing electrode earlier, a peak in the raw impedance signals in time, followed by the target droplets which are then screened in the upper channel by the other pair of sensing electrodes (Fig. 1 d and e). Consequently, the electrical impedance raw signals that present the negative and positive peaks caused by a pair of droplets are defined as single events. By extracting the peaks from the raw impedance signals, the processed impedance data can be delineated into clusters, wherein the cluster from the target droplets exhibits larger impedance values indicative of bacterial encapsulation, whereas the other, mixed with the reference droplets, represents empty droplets. The term “empty droplets” is meant to refer to droplets that do not contain any bacteria. It would be appreciated that target droplets that did not contain any bacteria has an impedance similar to that of the reference droplets. Calibrating the impedance data by dividing the peaks of target droplets byreference droplets in the respective events, a process termed auto-unbiasing dereferences the impedance data of empty droplets to 1. This enhances the consistency of droplet impedance measurements across different devices and experiments, ensuring a reliable evaluation of bacterial growth kinetics.

[0090] Example 2: Engineering phosphorylation-amplified bacterial culture medium

[0091] To foster bacterial metabolic activity, it is quintessential to formulate a culture medium endowed with both carbon and nitrogen sources. In this milieu, glucose is designated as the carbon source, being a non-ionizable organic compound, whilst peptone and yeast extracts serve as conductive nutritional mixtures, augmenting the medium's conductivity in direct proportion to their concentrations (R2= 0.99, Fig. 2a). Nevertheless, the medium's conductivity undergoes modulation owing to the hydrolytic byproducts of bacterial respiration, notably Trimethylamine, which engenders ionic hydroxide (Fig. 2b). This necessitates a meticulous calibration of the concentration of conductive nutrition to strike an equilibrium between conductivity amplification and bacterial growth sustenance.

[0092] As the change of conductivity in the bulk bacterial culture medium escalates with the augmentation of TMAO concentrations from 10 pl (0.833g / L with a conductivity of 699 pS / cm) to 60 pl (5g / L with a conductivity of 2376 pS / cm), a discernible retardation in growth is observed beyond 50 pl (4.167g / L) (Fig. 2c). The change of conductivity with addition of conductive nutrition from 10 pl to 100 pl (8.333g / L) of the original stock solution remains consistent in 20-hour incubation (about 2500 pS / cm). This may be because the incubation time is too long, and the conductivity of the bacterial culture medium has stabilized already. Then, the observation window is shortened to 5 hours, given the consistency of conductivity changes across varying conductive nutrition concentrations over the 20-hour incubation duration. Noteworthy, within the 5-hour incubation, the bespoke medium with an elevated conductive nutrition concentration (80 pl) mirrors a comparable bacterial growth rate to the conventional MH medium (Fig. 2d). However, the MH medium, albeit with identical TMAO concentrations as the customized medium, shows minimum change in conductivity, contrasting the customized medium which exhibits 2.3-fold and 3-fold amplifications with higher (80 pl) and lower (20 pl) conductive nutrition concentrations, respectively.

[0093] In the quest to optimize the conductive nutrition concentration, a thorough examination was conducted on the bacterial growth rate and the conductivity of the customized culture medium with diverse conductive nutrition additions (Fig. 2e). Although an escalation in conductive nutrition concentration propels enhanced bacterial growth, it concurrently attenuates the conductivity change rate, delineated as the ratio of conductivity at5 hours to that of the initial sample. Consequently, the optimized conductive nutrition concentration spectrum is demarcated between 20 to 60 pl of the original stock solution.

[0094] To substantiate the nexus between bacterial growth and culture medium conductivity, two samples bearing disparate initial bacterial concentrations were scrutinized (Fig. 2f). Irrespective of the initial bacterial concentration, the conductivity growth of the customized medium correlates with the bacterial concentration increment, albeit with a temporal lag in relation to bacterial growth. This temporal lag could be ascribed to the more pronounced TMAO consumption process ensuing post-substantial bacterial growth. It is noteworthy that a steady state is manifested during the initial 4-hour incubation in the sample with a lower initial bacterial concentration. This could be ascribed to the minuscule bacterial concentration, rendering it undetectable either by optical density or conductivity in bulk. Nonetheless, high linearity is retained between bacterial growth and the conductivity of the customized medium (Fig. 2g) for low (R2= 0.97) and high (R2= 0.91 ) initial concentrations (whereas is non-correlated for MH medium, R2= 10'4in Fig. 6), underscoring the robustness and viability of the phosphorylation-amplified bacterial culture medium in monitoring bacterial growth kinetics.

[0095] Example 3: Functional analysis of SYNC chip

[0096] In traditional single-channel impedance cytometry, the necessity for calibration microparticles arises to concurrently test with target samples, accounting for the potential variances in electrical signals emanating from device-to-device discrepancies and electronic wire connections. Calibration particles can either be amalgamated with target samples or characterized in advance of each experiment, engendering ambiguity in clustering identification due to sample admixture or sequential sample loading. While parallel cell screening has been posited to ameliorate this issue, the randomness and uncontrollability of events in electrical signals of target and reference remain a challenge attributed to the stochastic arrival of free-flow cells at the detection electrodes. Conversely, droplets traversing a microfluidic channel exhibit rhythmic flow governed by the injection pressure and intervening spacing oil, thereby harmonizing the generation frequency. This distinctive droplet behavior facilitates the synchronized pairing of target and reference droplets for electrical characterization. Despite the controllable injecting frequency, phase control presents a hurdle, potentially leading to missed droplet pairings. Consequently, a rail-based design is employed to actualize passive droplet self-synchronization (Fig. 3a). When droplets fully block the rails, the fluidic volume in front of the droplets are confined. When droplets are not synchronized, the connected channels between the rails help to balance the volume, achieving droplet paring (Fig. 7). Note that the sensing electrodes in the microfluidic channels are spaced 20 pm fromthe central electrode (power electrode). Therefore, to prevent disruption of the electrical signal, the maximum allowable mismatch between two adjacent droplets should preferably be no more than 20 pm. Upon injecting droplet pairs into parallel channels with identical frequencies, the channels, courtesy of multiple interconnectors, equilibrate the carrying oil volume between droplet pairs, thus achieving droplet synchronization.

[0097] As droplets traverse past the sensing electrodes, simulation outcomes elucidate that AC electrical current can permeate the oil barrier, interfacing the Au electrode and aqueous phase within droplets, thereby enabling the detection of aqueous phase conductivity. An elevated aqueous phase conductivity from 600 to 1200 pS / cm instigates a more pronounced alteration in electrical impedance signals (Fig. 3b). By extracting the peak of each event in electrical impedance signals, two samples comprising target droplets encapsulating solutions with conductivities of 70 and 60 pS / cm, and reference droplets encapsulating solutions with 60 pS / cm, are screened in SYNC (Fig. 3c and the detail single processing of SYNC can be found in ’’Operation of SYNC” under the Material and Methods section). Impedance data from target droplets bifurcate into two clusters (dark grey), among which, the cluster overlapping with reference data (light grey) is readily identifiable as low conductive droplets. Post auto-unbiasing of individual events, the impedance data of target droplets gravitate around a normalized value of 1 , aligned with the dotted reference line. Consequently, low conductive droplets, akin to reference droplets, exhibit a mean of 1 in impedance data, while high conductive droplets register impedance values exceeding 1 .

[0098] Further scrutiny of the SYNC system's detection limit is conducted (Fig. 3d). SYNC unveils an ultra-wide detection range, corroborating droplet conductivity spanning from 23 to 1600 pS / cm (R2= 0.99) whilst preserving high linearity within the interest region of 700 to 1200 pS / cm (R2= 0.97) with the resolution of ~100 pS / cm. This implicates SYNC's aptitude in furnishing accurate droplet conductivity measurements, which is pivotal for biomedical applications.

[0099] Example 4: Amplified detection of bacterial growth kinetics

[0100] Monitoring bacterial growth kinetics with rapid turnaround times is imperative across various disciplines, aiding timely interventions in healthcare, ensuring food and water safety, and bolstering microbiological research. Traditionally, plate counting, regarded as the gold standard due to its high accuracy, is employed for monitoring bacterial concentrations. However, it necessitates cumbersome sample dilution steps and a lengthy 24-hour incubation period to yield results. Alternatively, optical density furnishes an expedient method for realtime monitoring of bacterial growth kinetics, albeit its limited detection threshold engenders inaccuracies when examining low-concentration bacterial samples.

[0101] Addressing these constraints, the SYNC system amalgamates droplet microfluidics and electrochemical amplification, thereby rendering a viable solution for bacterial growth kinetics monitoring, particularly in samples with low bacterial concentrations (Fig. 4a). The accuracy of SYNC is assayed by evaluating bacterial samples with a high initial concentration (OD6OO = 0.2). Following bacterial encapsulation into droplets, multiple bacteria are observed within single droplets due to the high initial concentration (0 hr microscopic image, Fig. 4b). Monitoring the preset target gate in the impedance scatter, designated for measuring droplet conductivity encapsulating bacteria, reveals an augmentation in both data points and average impedance value within the target gate (top, Fig. 4b). This observation is corroborated by 3- hour and 5-hour incubation microscopic images, eventually discerning an isolated cluster indicative of bacterial-encapsulated droplets from the original cluster representing empty droplets. Counting the SYNC impedance data points (SYNC count) within the target gate to ascertain bacterial growth within individual droplets demonstrates a similar growth rate (slope k = 0.23) to optical density methods (k = 0.22), whereas the average impedance value within the target gate exhibits a diminished accuracy in bacterial growth measurement within droplets (k = 0.15). SYNC count also manifests a good linearity with optical density measurements (R2= 0.88 in Fig. 4d and R2= 0.98 in Fig. 10).

[0102] In bulk bacterial suspension, the volume ratio of total bacterial volume to sample volume diminishes linearly with declining bacterial concentration, complicating bacterial growth measurement (Fig. 5a). Contrarily, the picoliter confinement of microdroplets establishes isolated microenvironments for individual bacteria, maintaining a constant volume ratio of 1.56 x 10'5when bacterial concentration descends below 106bacteria / ml. Yet, low bacterial suspension concentrations pose a challenge of low probability single-bacteria encapsulation (less than 0.6% of singlets, Fig. 5b). Herein, the highly sensitive SYNC system proves invaluable for scrutinizing the scant number of bacterial singlets. Employing an initial concentration of 5x104bacteria / ml for bacterial encapsulation, single bacteria are encapsulated within individual droplets (left microscopic image, Fig. 5c). A 5-hour incubation period reveals an escalation in bacteria-encapsulated droplet density, mirrored by an increase in the number of SYNC impedance data points within the preset target gate for both E. coli (Fig. 5c and Fig. 8) and K. Pneumoniae (Fig. 9a). Validating bacterial concentration at each 1-hour interval during the 5-hour incubation using gold standard plate counting, SYNC unveils a limit of detection (LOD) for measuring bacterial growing from ~ 2.5x105bacteria / ml to ~ 6.6x105bacteria / ml, exhibiting a -5-fold enhancement over the OD600 approach (detectable from ~ 1.3x106bacteria / ml to - 2.6x10sbacteria / ml), thereby affirming prowess of SYNC in monitoring bacterial growth kinetics in low concentration samples, which remain elusive forconventional optical methods (Fig. 5d). Owing to the heightened sensitivity in bacterial growth detection, SYNC commences bacterial growth detection at 2 hours for E.coli and 1 hour for K. Pneumoniae, a 50% time reduction compared to the OD600 approach, which attains statistical significance at 4 hours and 2 hours respectively (Fig. 5e and Fig. 9b). This underscores aptitude of SYNC to accurately deduce bacterial growth kinetics parameters such as growth rate and bacterial doubling time, areas where conventional optical density methodology falters.

[0103] The initial bacterial concentration in suspension samples is a pivotal parameter in delineating bacterial growth kinetics. Regrettably, to the present inventors’ knowledge, existing real-time monitoring platforms fall short of measuring initial concentrations, typically assessing relative alterations in electrochemical signals. SYNC diverges by employing droplets to encapsulate single bacteria, thereby enabling computation of initial bacterial concentrations from the number of droplets encapsulating single bacteria, divided by the product of individual droplet volume and the total number of screened droplets. Hence, examining different incubation time steps ranging from 1 to 5 hours, a 4-hour incubation period suffices to ascertain the initial sample concentration (p-value> 0.5), aligning with results procured from plate counting from 24-hour incubation (Fig. 5f).

[0104] While embodiments of the invention have been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

CLAIMS1 . A method for determining a bacterial concentration in a sample, the method comprising the steps of:(a) introducing (i) a target droplet comprising the sample and (ii) a reference droplet comprising a control medium into an input region of a microfluidic device, the input region comprising a first input channel and a second input channel, wherein the target droplet is introduced into a carrier fluid in the first input channel and the reference droplet is introduced into the carrier fluid in the second input channel;(b) flowing the target and reference droplets from the input region into a synchronisation region to synchronise a pairing of the target and reference droplets;(c) flowing the target and reference droplets from the synchronisation region into a detection region to obtain a raw impedance signal for the pair of target and reference droplets;(d) calculating a calibrated impedance value by dividing a peak impedance value of the raw impedance signal corresponding to the target droplet with a peak impedance value of the raw impedance signal corresponding to the reference droplet; and(e) determining the bacterial concentration from the calibrated impedance value.

2. The method of claim 1 , wherein the sample comprises bacteria suspended in a culture medium.

3. The method of claim 1 or 2, wherein the control medium comprises a culture medium.

4. The method of claim 2 or 3, wherein the culture medium comprises peptone, yeast extract, trimethylamine N-oxide (TMAO) and glucose.

5. The method of claim 4, wherein the peptone is present at a concentration of between 0.6 and 5.4 g / L of the culture medium.

6. The method of claim 4 or 5, wherein the yeast extract is present at a concentration of between 0.3 and 2.7 g / L of the culture medium.

7. The method of any one of claims 4 to 6, wherein the TMAO is present at a concentration of between 0.833 and 5 g / L of the culture medium.

8. The method of any one of claims 4 to 7, wherein the glucose is present at a concentration of between 75 and 150 g / L of the culture medium.

9. The method of any one of claims 1 to 8, wherein step (a) comprises forming the target and reference droplets directly into the carrier fluid in the input region.

10. The method of any one of claims 1 to 8, wherein step (a) comprises pre-forming the target and reference droplets and subsequently introducing the pre-formed target and reference droplets into the carrier fluid in the input region.

11. The method of any one of claims 1 to 10, wherein the target and reference droplets are formed via an encapsulation method selected from the group consisting of microfluidic emulsion generation, vortexing and pipetting.

12. The method of any one of claims 1 to 8 or 10 to 1 1 , wherein the pre-formed target and reference droplets are incubated for a predetermined duration before being introduced into the carrier fluid in the input region.

13. The method of claim 12, wherein the predetermined duration ranges from 1 to 5 hours.

14. The method of any one of claims 1 to 13, wherein step (e) comprises counting a number of the calibrated impedance values falling within a predetermined impedance range, wherein a higher number of calibrated impedance values falling within the predetermined impedance range is indicative of a higher bacterial concentration in the sample.

15. The method of any one of claims 1 to 13, wherein step (e) comprises averaging the calibrated impedance values falling with a predetermined impedance range, wherein a higher average value of the calibrated impedance values falling within the predetermined range is indicative of a higher bacterial concentration in the sample.

16. The method of any one of claims 1 to 15, wherein the microfluidic device is the microfluidic device of any one of claims 17 to 21 .

17. A microfluidic device comprising: an input region comprising a first input channel and a second input channel, the first input channel being configured to receive a target droplet and the second input channel being configured to receive a reference droplet; a synchronisation region comprising a first synchronising channel and a second synchronising channel, the first synchronising channel being in fluid communication with the first input channel and configured to receive the target droplet from the first input channel, the second synchronising channel being in fluid communication with the second input channel and configured to receive the reference droplet from the second input channelwherein the synchronisation region is configured to synchronise the pairing of the target and reference droplets; a detection region comprising:(i) a first detection channel and a second detection channel, the first detection channel being in fluid communication with the first synchronising channel and configured to receive the target droplet from the first synchronising channel, the second detection channel being in fluid communication with the second synchronising channel and configured to receive the reference droplet from the second synchronising channel; and(ii) an electrode arrangement configured to measure an impedance of the target and reference droplets.

18. The microfluidic device of claim 17, further comprising a means for introducing the target droplet and reference droplet into the input region.

19. The microfluidic device of claim 17 or 18, wherein the first and second synchronising channels are interconnected in a rail-like arrangement in which the first and second synchronising channels have substantially perpendicular interconnecting passages therebetween to allow fluid communication between the first and second synchronising channels, the interconnecting passages being configured to allow a carrier fluid to pass between the first and second synchronising channels, wherein the rail-like arrangement is configured to synchronise the pairing of the target and reference droplets.

20. The microfluidic device of any one of claims 17 to 19, wherein the first detection channel and the second detection channel are substantially parallel to each other.

21. The microfluidic device of any one of claims 17 to 20, wherein the electrode arrangement comprises a central electrode, a first sensing electrode and a second sensing electrode, wherein the central electrode, the first sensing electrode and the second sensing electrode are substantially perpendicular to the first and second detection channels, wherein the first and second sensing electrodes are disposed on opposite sides of the central electrode such that the first sensing electrode, the central electrode and the second sensing electrode are substantially parallel to one another, wherein the first sensing electrode and the central electrode are configured for measuring an impedance of the plurality of reference droplets and wherein the central electrode and the second sensing electrode are configured for measuring an impedance of the plurality of target droplets.

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