WO2025233956A1

WO2025233956A1 - An integrated lab on chip device for the isolation, lysis and detection of pathogen from body fluids

Info

Publication number: WO2025233956A1
Application number: PCT/IN2025/050545
Authority: WO
Inventors: Sushama Agarwalla; Suhanya Duraiswamy
Original assignee: Indian Institute of Technology Hyderabad
Current assignee: Indian Institute of Technology Hyderabad
Priority date: 2024-05-07
Filing date: 2025-04-04
Publication date: 2025-11-13
Anticipated expiration: 2026-11-07

Abstract

The present invention discloses a microfluidic chip device for isolating a pathogen from body fluid. The present invention also relates to a process for isolating a pathogen from the body fluid using the microfluidic chip device.

Description

AN INTEGRATED LAB ON CHIP DEVICE FOR THE ISOLATION, LYSIS AND DETECTION OF PATHOGEN FROM BODY FLUIDS

FIELD OF THE INVENTION

The present invention relates to a device and method for isolation of pathogen from body fluids. The present invention also relates to an integrated microfluidic chip device for isolating pathogen from whole blood.

BACKGROUND OF THE INVENTION

Pathogen identification and detection are crucial for timely and effective treatment of infectious diseases like sepsis, which can be life-threatening and present symptoms similar to non-emergency bacterial infections. Currently, gold standards for disease detection involves culturing the pathogen in selective growth media, which typically takes 1 to 2 days to get detectable concentrations of pathogen [1]. Kits like SeptiFast (Roche) and SepsiTest (Molzyme Molecular Diagnostics) use nucleic acid-based techniques (NAT) to identify specific pathogen from blood, even non-cultivable and nonviable ones, provided the pathogen concentration is sufficient for detection.

However, eukaryotic DNA, such as cell-free DNA and DNA from white blood cells (WBCs), and proteins present in high concentrations in the blood can interfere with and inhibit PCR reactions, affecting the sensitivity and selectivity of these methods [2, 3]. Pretreatment strategies that lyse eukaryotic cells and denature DNA and proteins in the blood are often used, but the chemicals used can inhibit PCR and require further purification steps [2, 4, 5],

There are significant literatures reported for detection of sepsis causing pathogen based on different methods including NAT based approaches [6] as discussed in Table 1.

Table 1: Emerging molecular diagnostics for pathogen detection from whole blood [1]

However, these toolkits have several limitations, including:

• Sample Preprocessing Time: The preprocessing time for samples is not included in the toolkit, which can affect the overall efficiency and turnaround time of the diagnostic process.

• Negative Enrichment: Negative enrichment methods, while useful for removing background noise, can also result in the loss of pathogenic DNA, reducing the sensitivity of the assay. • Time-consuming Process: The overall process, including sample preparation, enrichment, and detection, can be time-consuming, limiting its utility for rapid diagnosis.

• Inhibition of Downstream PCR: Current pathogen enrichment strategies, while effective in isolating pathogen, can also inhibit downstream PCR reactions, affecting the accuracy and reliability of the assay.

Several microfluidic techniques such as Immunoaffinity, droplet-based, and inertial microfluidics and many more (mentioned in the Table 2) are also commonly employed for the isolation and detection of pathogen.

Table 2: Pathogen isolation from whole blood

Despite their effectiveness, these microfluidic techniques are associated with several disadvantages as below:

• Microfluidic Channel Clogging: Microfluidic channels can become clogged, leading to reduced efficiency and reliability of the pathogen isolation and detection process.

• Non-label Free: Some techniques require labeling of pathogen with fluorescent or other markers, which can be time-consuming and may affect pathogen viability.

• Degradation of Reagents: Reagents used in microfluidic techniques can degrade over time, diminishing the sensitivity and accuracy of pathogen detection. • Multiple Sample Processing Steps: Certain techniques necessitate multiple processing steps prior to detection, increasing the complexity and time required for analysis.

• Most of these methods either perform pathogen isolation or detection individually.

Immunoaffinity -based methods offer high specificity but come with high costs and potential antibody instability. Wang et al. achieved a 70.7% capture efficiency for E. coli with a limit of detection of 50 cells/mL, but the method's effectiveness with gram-positive bacteria is unknown.

Bisceglia et al. demonstrated promising results in capturing E. coli and other pathogens using dielectrophoresis. However, the method's complexity and the need for careful parameter optimization may limit its practicality. The minimum cell per load required for detection is 10⁴ CFU/mL.

Cai et al. created a microfluidic device that combines dielectrophoresis with on-chip multiplex array PCR, achieving high capture efficiencies for E. coli. However, its ability to isolate other pathogens, especially gram-positive bacteria, was not reported. The minimum cell load required for detection is 10³ CFU/mL.

Cooper et al. developed a micro-device that uses immunomagnetic isolation followed by on- chip optical detection. While they achieved a high capture efficiency of 98% for C. albicans fungi, the method has not been tested for gram-negative and gram-positive bacteria.

Fang et al. presented an integrated microfluidic device capable of removing white blood cells and red blood cells, as well as capturing bacteria using magnetic beads. While the method showed high capture rates, further optimization of the filtering and capture rates may be necessary for improved efficiency, especially with different bacterial strains.

Therefore, there is a need in the art to address these limitations and to provide a device and method having enhanced efficiency and applicability of microfluidic techniques in pathogen isolation and detection.

OBJECTIVE OF THE INVENTION

An objective of the present invention is to provide a microfluidic chip device which is capable of performing isolation and lysis of pathogen on a single platform.

Another objective of the present invention is to provide microfluidic chip device which is capable of performing isolation, lysis and detection of a pathogen on a single platform.

Still another objective of the present invention is to provide a method for isolation and lysis of a pathogen using a microfluidic chip device. Yet another objective of the present invention is to provide a method for isolation, lysis and detection of a pathogen at low concentration of pathogen in the body fluid using a microfluidic chip device.

These and other objects and advantages of the present subject matter will be apparent to a person skilled in the art after consideration of the following detailed description taking into consideration accompanying drawings in which preferred embodiments of the present subject matter are illustrated.

SUMMARY OF THE INVENTION

An aspect of the present invention provides a microfluidic chip device comprising:

(a) an upper layer (layer 1);

(b) an intermediate layer (layer 2) comprising a plurality of inlets (inlet 1, inlet 2, inlet 3), an outlet 1, a plurality of microfluidic channels and a plurality of wells (Wl, W2, W3); and

(c) a lower layer (layer 3) comprising a plurality of wells (W4, W5, W6) and an outlet 2; wherein the microfluidic channels are in connection with inlet 1, inlet 2, inlet 3, the plurality of wells (Wl, W2, W3) and outlet 1.

Another aspect of the present invention provides a method for isolating a pathogen from a body fluid sample comprising: i. introducing the body fluid sample via inlet 1, a gas via inlet 2 and protein coated magnetic beads with buffer via inlet 3 into a microfluidic chip device; ii. maintaining the sample flow rate in the range of 1 pL/min to 10 pL/min and gas pressure in the range of 20 psi and 30 psi in microfluidic channels; iii. allowing capture of pathogen attached to the protein coated magnetic beads in the plurality of wells (Wl, W2, W3); iv. placing a magnet beneath the plurality of wells (W4, W5, W6) to attract pathogen attached to the magnetic beads; v. collecting pathogen attached to the magnetic beads from outlet 2; vi. separating the pathogen from the magnetic beads; and vii. collecting waste sample from outlet 1.

These and other aspects of the disclosed subject matter, as well as additional novel features, will be apparent from the description provided herein. The intent of this summary is not to be a comprehensive description of the claimed subject matter, but rather to provide a short overview of some of the subject matter's functionality. Other systems, methods, features and advantages here provided will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features and advantages that are included within this description, be within the scope of any claims.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The illustrated embodiments of the subject matter will be best understood by reference to the drawings. The following description is intended only by way of example, and simply illustrates certain selected embodiments of composite and processes that are consistent with the subject matter as claimed herein, wherein:

Figure 1 shows the schematic of segmented microfluidics.

Figure 2 shows schematic of the microfluidic chip device experimental setup.

Figure 3 Bar chart showing the concentration of bacteria in spiked blood along with the concentration of bacteria grown when the beads are spread onto agar plates after mixing, incubation and magnetic separation using the base manufacturer’s protocol and modifications specified on the X-axis.

Figure 4 shows plots of the mixing efficiency against the total liquid flow rate at different pressure in microfluidic chip device of different designs shown in the respective insets (a) Design 1 (DI) (b) Design 2 (D2) (c) Design 3 (D3) (d) Comparing three different designs at optimum pressure with different flowrate. The original concentration of bacteria spiked in blood was IxlO⁴ CFU/mL and the concentration of the beads in the buffer was 4xl0⁹ beads/mL for all experiments conducted. Figure 5 shows schematic of the different layers that are aligned parallelly and bonded to form the integrated microfluidic chip device to facilitate gas separation and isolate pathogens from whole blood. The mixing zone containing the D3 design forms layer 2 while the magnetization zone containing three wells (W4, W5, W6) to capture the beads-pathogen complex from the segmented microflow unit forms layer 3 which also contains the inlet and outlet ports. The separated gas is allowed to escape through the filters placed on layer 1.

Figure 6 shows schematic of the integrated microfluidic chip device the three layers aligned parallelly and bonded together.

Figure 7 is the schematic of the experimental setup for pathogen sequestering in the segmented flow zone and their isolation by magnetization when “Magnet on” condition. The segmented blood flows through the microchannel (layer 3) forming the beads pathogen complex which are then collected in the 3 wells in layer 3. When the magnet is turned on, the beads pathogen complex is pulled down to the bottom of the well, while the waste blood is directed towards the outlet 1. The lighter gas in the segmented blood is allowed to escape through the layer 1 through the flow membrane.

Figure 8 is the schematic of the experimental setup for pathogen sequestering in the segmented flow zone and their isolation by magnetization when “Magnet off’ condition. The isolated beads pathogen complex in the well is collected by-passing PBS buffer through the inlet port. The collected beads pathogen complex is then spread onto agar plates to determine the concentration of pathogen sequestered.

Figure 9 shows schematic of the experimental setup illustrating the pathogen isolation process after the magnetization is turned off, showing beads pathogen complex, passed on to the micro-piezo actuator device (TSAW On) for lysis of the pathogen. Subsequently, the lysate is analyzed by qPCR for the detection of the pathogen.

DETAILED DESCRIPTION OF THE INVENTION

A detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such detail as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

The terminology used herein is to describe particular embodiments only and is not intended to be limiting to the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising”, or “includes” and/or “including” or “has” and/or “having” when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The term “further” is used in the embodiments and claims of the present application. The said term is a well-accepted term to narrow down any principal feature. Therefore, the person skilled in the art would clearly understand the scope of the said term in the context of the present disclosure.

In accordance with the present invention, a “pathogen” is an organism causing disease to its host, with the severity of the disease symptoms referred to as virulence. Pathogens are taxonomically widely diverse and comprise viruses and bacteria as well as unicellular and multicellular eukaryotes.

“Microfluidic Device,” as used herein, refers to a device that includes one or more microfluidic channels, one or more microfluidic valves, one or more microfluidic chambers, or combinations thereof, and are configured to carry, store, transport, and/or analyze samples in fluid volumes of less than ten milliliters (e.g., in fluid volumes of 5 mL or less, in fluid volumes of 2.5 mL or less, or in fluid volumes of 1.0 mL or less). The microfluidic device described herein can be configured to individually process a variety of samples. As used in this context, the term “process” can include transporting the individual members of one or more sample populations to a sample processing element (or fluid outlet), manipulating and/or interrogating the individual members of one or more sample populations, or combinations thereof. The device can be fabricated using photolithography, soft lithography and also using additive manufacturing - 3D printing or other metal, polymer or glass-based fabrication techniques. Microfluidic device can be made of any polymer or glass or metal or silicon with a transparent surface.

“Microfluidic channel,” as used herein, refers to a feature within a microfluidic device that forms a path, such as a conduit, through which one or more fluids can flow. In some embodiments, microfluidic channels have at least one cross-sectional dimension that is in the range from about 0.1 microns to about 10 millimeters (e.g., from about 1 micron to about 5 mm, from about 1 micron to 1 mm, from about 1 micron to about 750 microns, from about 1 micron to about 500 microns, from about 5 microns to about 500 microns, or from about 5 microns to about 150 microns).

The microfluidic channels can independently be linear in shape, or they can have any other configuration required for device function, including a curved configuration, spiral configuration, angular configuration, or combinations thereof. The microfluidic channels can be fabricated to have a variety of cross-sectional shapes, including but not limited to, square, rectangular, triangular (i.e., v-shaped), hemispherical, and ovular.

As used herein, “body fluid” can include any fluid obtained from a body of a patient, including, but not limited to, whole blood, cerebrospinal fluid, urine, bile, lymph, saliva, synovial fluid, serous fluid, pleural fluid, amniotic fluid, and the like, or any combination thereof.

The present invention is directed towards a microfluidic chip device comprising:

(a) an upper layer (layer 1);

(b) an intermediate layer (layer 2) comprising a plurality of inlets (inlet 1, inlet 2, inlet 3), an outlet 1, a plurality of microfluidic channels and a plurality of wells (W1, W2, W3); and

(c) a lower layer (layer 3) comprising a plurality of wells (W4, W5, W6) and an outlet 2; wherein the microfluidic channels are in connection with inlet 1, inlet 2, inlet 3, the plurality of wells (Wl, W2, W3) and outlet 1. In an embodiment of the present invention, there is provided a microfluidic chip device, wherein an upper layer (layer 1) is equipped with a membrane filter paper to eliminate gas and contamination.

In another embodiment of the present invention, there is provided a microfluidic chip device, wherein the inlet 1 is for introducing a sample, the inlet 2 is for introducing a gas, the inlet 3 is for introducing magnetic beads with buffer, and outlet 1 is for collection of waste sample.

In yet another embodiment of the present invention, there is provided a microfluidic chip device, wherein the outlet 2 is for collecting pathogen bound to the magnetic beads with buffer.

In still another embodiment of the present invention, there is provided a microfluidic chip device, wherein the plurality of wells (Wl, W2, W3) are for capture of pathogen and the plurality of wells (W4, W5, W6) in lower layer (layer 3) are aligned with the plurality of wells (Wl, W2, W3) in intermediate layer (layer 2).

In an embodiment of the present invention, there is provided a microfluidic chip device, wherein the microfluidic channels are squiggle in shape and have dimension of 300 pm.

In another embodiment of the present invention, there is provided a microfluidic chip device, wherein the device is fabricated using a substance selected from the group consisting of poly dimethyl siloxane, silicon, quartz, glass, poly methyl methacrylate, polycarbonate, and cycloolefin polymer.

In yet another embodiment of the present invention, there is provided a microfluidic chip device, wherein the device is fabricated using poly dimethyl siloxane.

Another embodiment of the present invention provides a method for isolating a pathogen from a body fluid comprising:

(i) introducing the body fluid sample via inlet 1, a gas via inlet 2 and protein coated magnetic beads with buffer via inlet 3 into a microfluidic chip device;

(ii) maintaining the sample flow rate in the range of 1 pL/min to 10 pL/min and gas pressure in the range of 20 psi and 30 psi in microfluidic channels;

(iii) allowing capture of pathogen attached to the protein coated magnetic beads in the plurality of wells (Wl, W2, W3); (iv) placing a magnet beneath the plurality of wells (W4, W5, W6) to attract pathogen attached to the magnetic beads;

(v) collecting pathogen attached to the magnetic beads from outlet 2;

(vi) separating the pathogen from the magnetic beads; and

(vii) collecting waste sample from outlet 1.

In still another embodiment of the present invention, there is provided a method for isolating a pathogen from a body fluid, wherein the method further comprises passing the separated pathogen through a micro piezo-actuator for lysis to release nucleic acid.

In an embodiment of the present invention, there is provided a method for isolating a pathogen from a body fluid, wherein the method further comprises conducting an assay on the nucleic acid to identify the pathogen.

In another embodiment of the present invention, there is provided a method for isolating a pathogen from a body fluid, wherein the assay is selected from the group consisting of a sequencing reaction and a polymerase chain reaction.

In yet another embodiment of the present invention, there is provided a method for isolating a pathogen from a body fluid, wherein the assay is a polymerase chain reaction.

In still another embodiment of the present invention, there is provided a method for isolating a pathogen from a body fluid, wherein the body fluid sample is selected from the group consisting of serum, whole blood, sweat, semen, cerebrospinal fluid, saliva, tears, urine, vaginal secretions, synovial fluid, pleural fluid, pericardial fluid, nasal fluid, gastric fluid, and breast milk.

In yet another embodiment of the present invention, there is provided a method for isolating a pathogen from a body fluid, wherein the body fluid sample is whole blood.

In an embodiment of the present invention, there is provided a method for isolating a pathogen from a body fluid, wherein the pathogen is selected from the group consisting of fungi, bacteria, virus or a combination thereof.

The present invention describes a method for isolating pathogen from a sample, preferably a body fluid, more preferably whole blood using a “direct pathogen isolation technique” with protein-coated magnetic beads, microfluidic mixing, and TSAW (Travelling Surface Acoustic Wave)-based micro-piezo actuator for lysis, followed by PCR for detection.

The “direct pathogen isolation technique” offers a promising solution to the limitations of existing molecular diagnostics for pathogen detection from whole blood. By utilizing protein-coated magnetic beads and microfluidic technology, this method enables selective pathogen sequestering without the use of harmful chemicals and significantly reduces processing time and complexity.

The invention integrates a microfluidic mixing strategy to efficiently isolate pathogen from blood samples using protein-coated magnetic beads. This approach significantly reduces isolation time and enhances separation efficiency, even with lower concentrations of beads. Additionally, a TSAW-based micro-piezo actuator is used for rapid lysis of isolated pathogen. This process preserves intracellular components crucial for subsequent end-point detection using PCR. Importantly, the method requires no prior treatment of whole blood.

The method utilizes segmented microfluidics to achieve quick mixing and contact between magnetic beads and pathogen, thus reducing the time and steps involved in the process. This approach improves separation efficiency while using lower concentrations of magnetic beads, minimizing the number of steps and reducing the time required for the entire process. It enables the rapid isolation of pathogen from large volumes (1 mL-5 mL) of blood within a few minutes using a microfluidic chip device.

The method for pathogen isolation, lysis, and detection using the microfluidic chip device focuses on three key components:

• Isolation: Employing protein coated magnetic beads for pathogen isolation from whole blood utilizing segmented microfluidic chip device for enhanced efficiency.

• Lysis: Utilizing an acoustofluidic TSAW device for bacteria lysis, ensuring efficient and rapid disruption of pathogen cells without compromising the intracellular component.

• Detection: Implementing end point off chip Polymerase Chain Reaction (PCR) for pathogen detection, providing a highly sensitive and specific method for identifying pathogen species.

The process includes: ■ Offering a microfluidic chip device that includes a mixing zone and a magnetizing zone;

■ Introducing protein-coated magnetic beads and a blood sample into the device and enabling the beads to attach to pathogen in the mixing zone;

■ Separation of the mixture from the magnetizing zone, where the magnetic beads selectively attach to pathogen in the blood;

■ Using a TS AW -based micro-piezo actuator to quickly break down the isolated pathogen (Indian Patent 202341040780 dated 15 June 2023, included herewith by reference);

■ Ensuring the protection of intracellular components for future PCR detection.

The efficiency and accuracy of pathogen isolation followed by lysis and end point detection methods are improved by this technology, which makes it possible to isolate pathogen from whole blood quickly and effectively without the need for previous treatment.

The microfluidic device effectively isolates, magnetizes, and separates pathogen from whole blood samples, with a particular emphasis on pathogen that cause sepsis. This method involves using protein-coated magnetic beads to specifically trap pathogen without the use of any harmful chemicals.

Figure 1 displays the schematic of the process, utilizing segmented microfluidic method. As illustrated in Figure 1, the process consists of two primary sections. Two key areas are the mixing and binding zone and the magnetizing zone. The magnetizing zone plays a critical role by serving as the location where the magnetic beads specifically bind to pathogen in the blood. The effectiveness of this attachment depends on the mixing efficiency in this area, which then impacts the binding and separation efficiency of the pathogen from other blood components. Nevertheless, achieving effective mixing poses a challenge because of the viscosity of blood and the existence of magnetic beads, which hinders the use of traditional magnetic mixers intended for mixing large solution volumes.

Using segmented microfluidics or gas-liquid flows allows for quick mixing (complete in about 800 ps) due to the fluid’s recirculatory motion within the liquid compartment. This method has been effectively utilized in the present invention to attach the beads to pathogen. This technique involves blood containing pathogen forming the continuous fluid, with air (N2 gas) introduced through a T-junction to create evenly sized and spaced gas bubbles. These gas bubbles divide the blood into separate compartments of liquid plugs, leading to recirculatory fluid motions that efficiently mix the liquid. The magnetic beads in buffer solution are added later, through another T-junction. The fast mixing resulting from the re- circulatory motion within the plugs facilitates improved and effective interaction between the pathogen and the magnetic beads. This method provides the benefit of rapid and consistent mixing within the liquid plugs as they flow between the gas bubbles. Downstream, protein-coated magnetic beads in a buffer solution are introduced through another T- junction. The rapid mixing within the liquid plugs enhances contact between the pathogen and the magnetic beads, promoting efficient binding. This microfluidic device enables effective isolation of pathogen from whole blood, enhancing the efficiency and speed of the process.

The micro-piezo actuator is used to lyse the isolated pathogen rapidly, preserving intracellular components for subsequent PCR detection. PCR is used to detect the presence of pathogen based on amplified DNA sequences.

Unlike traditional methods that rely on complex protocols and multiple reagents for pathogen isolation, the present method uses protein-coated magnetic beads to selectively capture pathogen directly from whole blood. This eliminates the need for extensive sample preparation and reduces the risk of contamination. Traditional magnetic bead-based methods often require lengthy incubation periods and manual mixing steps, leading to increased processing time. The microfluidic device of the invention incorporates efficient mixing techniques, ensuring rapid and uniform interaction between the magnetic beads and the pathogen, thereby reducing the overall processing time.

The use of TSAW-based micro-piezo actuators for cell lysis enables rapid and effective lysis of captured pathogen. This method is gentler than traditional mechanical or chemical lysis methods, reducing the risk of damage to the target molecules and increasing the efficiency of downstream analysis. The use of endpoint PCR for pathogen detection offers high specificity and sensitivity, allowing for the reliable identification of sepsis-causing pathogen. This approach eliminates the need for costly and time-consuming sequencing techniques, making the overall process more efficient and cost-effective.

The continuous flow-based segmented microfluidic chip device offers fast and reproducible mixing within the liquid plugs flowing between the gas bubbles, providing a robust solution for pretreatment of blood samples in NAT -based pathogen isolation. This advancement is significant in medical diagnostics, offering an improved, efficient, and less labour-intensive method for the isolation, magnetization, and separation of pathogen from whole blood, followed by lysis and endpoint detection using PCR.

In diagnostic centers, this microfluidic chip device and method eliminates the need for preprocessing patient samples, allowing for direct pathogen isolation and subsequent lysis for pathogenic DNA extraction. The micro-piezo actuator enables rapid lysis of various pathogen within seconds (~4s), while preserving intracellular components crucial for subsequent end-point detection using PCR. This utility underscores the transformative impact of this invention in advancing diagnostic capabilities for sepsis and related infections. The microfluidic chip device extends to healthcare settings, benefiting clinical personnel and diagnostic laboratories by streamlining the diagnosis process for sepsis-causing pathogen. By reducing both time and procedural steps, this approach offers a more efficient method for pathogen isolation and identification. Thus, this invention represents a significant advancement in the field of molecular diagnostics, offering a more efficient and reliable method for pathogen detection that has the potential to revolutionize diagnosis and treatment of infectious diseases.

In summary, the technical effects of the invention that enhance its efficiency and effectiveness in isolating, lysing and detecting pathogen are:

• Utilizing protein-coated magnetic beads in the microfluidic chip device enables precise capture of pathogen from challenging samples such as whole blood. This enhances the sensitivity and precision of the isolation procedure.

• By incorporating the effective mixing techniques and quick lysis method, the total processing time is significantly reduced in comparison to conventional methods. It is essential for ensuring prompt diagnosis and treatment of sepsis.

• With its high sensitivity and specificity, the assay effectively captures specific pathogen, lyses them efficiently, and detects them through PCR, making it ideal for identifying sepsis-causing pathogen.

• The method for isolation of pathogen using the microfluidic chip device provides a solution for pathogen isolation and detection that is cost-effective by minimizing reagents and processing steps, thus increasing accessibility in resource-limited settings.

• The design of the microfluidic chip device makes it suitable for automation, which could lead to efficient processing of samples in clinical and research environments.

ADVANTAGES OF THE PRESENT INVENTION

■ The microfluidic chip device results in reduction in the amount of time and effort that is necessary for analysis of a pathogen.

■ High sensitivity and specificity in the identification of bacterial species can be achieved through the utilization of microfluidic chip device for isolation of pathogen, TSAW for the purpose of lysis, and PCR for the purpose of detection.

■ The microfluidic chip device can detect sepsis causing bacterial pathogen in a short amount of time, which is essential for an accurate diagnosis and prompt treatment in healthcare settings.

■ The microfluidic chip device can be utilized in a variety of healthcare and research settings, including point-of-care applications, because it consolidates several steps onto a single platform. This makes it easier to use.

■ The integrated design of the microfluidic chip device makes it possible to automate the detecting process of a pathogen, which eliminates the need for human interaction and reduces the likelihood of making mistakes.

■ The capability of the device to execute numerous activities on a single platform lowers the need for separate equipment and reagents, which may result in a reduction in overall costs.

■ This microfluidic chip device offers a substantial breakthrough in bacterial isolation, which can be further lysed after isolation and end point detection technology, with benefits in terms of efficiency, accuracy, speed, accessibility, automation, and costeffectiveness. In general, the use of this device offers a considerable advancement.

EXAMPLES

Following examples are given by way of illustration, therefore, should not be construed to limit the scope of the invention. Example 1

Standardizing parameters

To establish the baseline performance of the experiments, initial step involved following the manufacturer's protocol in flask-based studies. Subsequently, the protocol was customized to optimize parameters such as buffer volume, concentration, bead volume, and incubation time for maximum pathogen isolation from blood. Each parameter was systematically varied, and the outcomes were analysed before adjusting other parameters.

The modified base protocol included mixing equal volumes of spiked blood and buffer premixed with beads, followed by incubation on a thermomixer at 37°C and 1000 RPM for 30 minutes. The mixture was then subject to magnetic separation on a magnetic stand for 10 minutes to isolate the beads, after which the supernatant was removed. The collected beads were dispersed in 100 pL of IX PBS for concentration determination. This process was repeated five times, and the results were plotted in Figure 3. The capture efficiency, as depicted in Figure 3, was approximately 90%. Further optimization of the base protocol was conducted to explore the maximum tunability of parameters for the development of the microfluidic setup.

Figure 3 illustrates the corresponding capture efficiencies when varying the buffer concentration to 4X, adjusting the temperature to room temperature, reducing the bead volume from 20 pF to 10 pL, and decreasing the mixing and incubation times to 20 and 10 minutes, respectively. The capture efficiencies for these variations were 7.64%, 80.33%, 40.39%, 82%, and 79.21%, respectively. Concentrating the buffer and reducing the bead concentration in the reaction mixture led to lower bacteria capture rates, while the other parameters tested had an insignificant effect.

This suggests that the incubation time can be reduced to 10 minutes, and the operating temperature does not need to exceed room temperature. These conditions were directly applied in the design of the cartridge and the segmented microfluidic chip.

Example 2

Designing and fabricating microfluidic chip device The microchannels were fabricated using poly (dimethyl siloxane) (PDMS). The microchannels were conceptualized and designed using Autodesk fusion 360. Further the microchannels were converted to. bff from .stl file and printed using SLA based 3D printer (ProJet 600, 3D System, USA). The channels feature a rectangular cross-section, measuring 300 pm in width and approximately 150 pm in depth. Channel patterns are molded and sealed onto glass slides that have been pre-coated with a thin layer of cured PDMS following a 45-second air plasma treatment to obtain a microfluidic chip.

Example 3

Experiments using microfluidic chip device

Experiments using the microfluidic chip device involved several key steps and considerations. Human blood was obtained from a healthy volunteer at the Indian Institute of Technology Hyderabad clinic, Kandi, Sangareddy, Telangana - 502284, India with proper consent and supervision. This human blood was spiked with known concentration of bacteria (Escherichia coli, DH5 alpha (E. coli ATCC 10798), Staphylococcus aureus (S. aureus ATCC 6538). Magnetic beads (ApoH lipoprotein coated magnetic beads, 200 nm diameter) are from ApoH Manufacturers in France.

The experimental setup, as shown in Figure 2, include the overnight culturing of the bacteria in LB cultures (aerobic, non-stirring conditions) at 37 °C. In the experimental set-up, the concentration of bacteria varied from 10⁵ CFU/mL to 10 CFU/mL and they were then used to spike the blood sample, and the mixture was thoroughly mixed.

1 mL of Spiked blood (with bacteria concentration varying from 10⁵ CFU/mL to 10 CFU/mL) and magnetic beads (20 pL of ApoH beads), mixed in 1 mL of 2X buffer solution [composition of 10X buffer solution: Tris-HCl pH8.0 IM; NaCl 1.5 M; Glycine lOOmM; BSA (Bovine Serum Albumin) 10% (w/v); from 10X buffer, 2X buffer was prepared), were delivered into individual T-junctions using separate syringe pumps (Fusion 101, Chemyx Inc., USA).

Inert gas (N2) or air from a cylinder equipped with a two-stage pressure regulator was delivered into the microfluidic chip gas inlet and pressure was controlled with a pressure controller (Line up flow EZ-2000). The flow rates of the blood, magnetic beads, and gas, along with the gas pressure, were carefully controlled to create segmented flows in the microfluidic chip. It was crucial to maintain these segmented flows at a level that allowed for contact between the pathogen/bacteria and the magnetic beads without causing detachment of the formed complex due to high mixing shear.

Example 4

Blood plug and gas bubble size optimization

The size of blood plugs is crucial for effective mixing in microfluidic systems. Studies have shown that plug with a length approximately equal to two widths of the channel result in optimal mixing [34].

The experiments involved measuring plug size and gas bubble formation under various conditions, including blood flow rates ranging from 1 pL/min to 30 pL/min and gas pressures from 10 psi to 30 psi.

It was observed that at gas pressures below 20 psi, the appropriate size of blood plugs could not be achieved, leading to a flipping region that hindered mixing efficiency. Larger plugs exhibited a flipping phenomenon, where the back of the plug moved from one side to another, whereas smaller plugs showed more efficient mixing without flipping [34, 35].

Quantifying mixing in microfluidic systems presents challenges such as over-twirling and incomplete snap-off, which can disrupt the time-distance relationship. Over-twirling causes mixed solution to enter the aqueous inlet of the plug-forming region, while incomplete snap- off leaves a portion of the solution at the inlet, leading to an apparent and incorrect mixing time of zero.

For gas pressures between 20 psi and 30 psi and flow rates ranging from 1 pL/min to 10 pL/min, the formation of gas segments length (~ 0.2 to 2.5 mm) between adjacent liquid plugs was ensured. This promoted efficient recirculatory mixing within each plug. However, at a channel dimension of 300 pm, a flow rate of 30 pL/min could not be exceeded due to pressure drop, leading to improper plug size. This limitation is likely caused by factors such as channel length, channel geometry, fluid viscosity, and flow rate, which affect the formation and stability of the plugs. While testing higher flow rates at larger channel dimensions (600 m), still desired plug size for effective mixing could not be achieved. These findings are important considerations for future mixing studies in microfluidic systems.

Example 5

Microfluidic Mixing Studies

Segmented microfluidic flows were generated using T-junction geometry with the blood spiked with pathogen/bacteria (10⁴ CFU/mL) flowing in as the continuous fluid and gas as the dispersed phase fluid (Figure 1).

The blood is hence compartmentalized into nanolitre volume plugs by the gas bubbles into which beads are added using a downstream inlet. Mixing was effected using recirculatory fluid motion along the top and bottom halves of the liquid plug thus enabling the contact between the bacteria and protein coated magnetic beads.

Three different channel designs were used to test the mixing efficiency and the results are shown in Figure 4. Mixing in segmented microfluidics depends on several factors such as (1) the length of the fluid plug, which dictates the recirculation efficiencies; (2) gas pressures and liquid flow rate which dictates the residence time of the fluids in the reactor; and (3) the reactor design - straight channel against the serpentine channel which provides the curvature required to stretch and fold the fluid leading to chaotic advection within the liquid plugs.

The baker's transformation was employed as a reference to simulate the chaotic advection within plugs during the mixing process. This process entails a sequence of stretching, folding, and reorienting occurrences that result in a significant reduction in the thickness of striations, following an exponential pattern. This procedure is comparable to the idea of a “rolling droplet”. These include recirculating flow in straight sections, which leads to folding and stretching, as well as reorientation at turns. The striation thickness diminishes dramatically with each cycle. The configuration of the channel impacts the recirculating flows and can be detected by examining the unevenness of the vortices within the plug. Vortices in a straight channel exhibit symmetry, whereas in a smooth turn, they display asymmetry. After making a sudden turn, there is a prominent vortex that symbolizes the realignment of the plug. Therefore, to study the effect of channel geometry, straight channel was designed (Design 1:D1), and designs with different number of channel turns (squiggle in between straight channel: Design 2, D2) to complete squiggles (Design 3, D3).

Design 1 (DI): straight channel, did not lead to complete mixing, because mixing in straight channels confined recirculations to only the upper and lower halves of the droplets i.e. the mixing efficiency in a straight microchannel diminishes in an inverse relationship with the diameter of the channel when a plug of fluid passes through it (Figure 4a). The decrease in efficiency is caused by the fluid’s motion direction in relation to the walls, resulting in the formation of two vortices within the plug instead of just one. Although the vortices aid in blending the substances within each section of the plug, there is no transfer of fluid between the two sections, resulting in inadequate total mixing.

The scaling argument for chaotic mixing in microchannels [30], which draws inspiration from the baker's transformation [31], is based on prior research in chaotic mixing. Two fundamental assumptions were established. Initially, it was hypothesized that during one cycle of chaotic advection, a plug would need to cover a distance equivalent to a specific multiple of its own lengths, which is directly related to the cross-sectional size of the microchannel. This premise is valid because as channels decrease in size, it results in smaller plugs. Furthermore, it was postulated that the mixing time corresponds to the point at which the durations of convective transport and diffusive mixing are in equilibrium, which is roughly equivalent to the overall residence time.

By incorporating this concept into the equation for mixing only through diffusion, the timescale for diffusion-based mixing after n cycles was derived [32]. An estimation was made for the duration required for convection to complete n cycles of transportation. Based on the second assumption, the mixing time was determined as the moment when the rate of mixing through diffusion becomes equal to the rate of transport through convection. This approach offers valuable insights into the phenomenon of mixing in microchannels and enhances the understanding of fluid flow dynamics at small scales.

An argument based on scaling was developed to elucidate the relationship between the mixing time in microfluidic systems experiencing chaotic advection and the channel width, flow velocity, and fluid diffusion coefficient. This reasoning assumed that during a single occurrence of chaotic advection, a plug must travel a distance equal to a specific multiple of its own length, and that the time it takes for mixing to happen is approximately when the durations of convective transport and diffusive mixing coincide. The theory stated that the mixing time exhibits a logarithmic relationship with the Peclet number (Pe), which quantifies the ratio between advection and diffusion rates.

The mixing time, t_m, is defined as the point at which the timeframe for mixing by diffusion is equal to the timescale for transport by convection [30]: where, t_convection time-scale for transport by convection n is the number of folds, stretch and reorient cycles l_p length of the plug w width of the microchannel v flow velocity o factor that influences the cycle of chaotic advection is the decrease in striation thickness.

D diffusion coefficient

^diffusion time-scale for transport by diffusion

After rearrangement of equation 1

As it is known,

The value of n is obtained by applying the logarithm function to both sides of the equation

(2) and assuming large values of the Peclet number: n~log (Pe) (4) In microfluidic design for mixing applications, the number of squiggles (n) plays a crucial role, particularly in microchannels where the Peclet number is significant. The value of n is directly related to the width of the channel and the flow velocity, and inversely related to the diffusivity coefficient.

For the present invention involving blood flow rates less than 10 pL/min and gas pressure between 20 psi to 30 psi, a critical condition occurs where the plug size is smaller than the bubble size. This condition is crucial for efficient mixing. Specifically, for a blood flow rate of 5 pL/min, the corresponding n value is approximately 8. Similarly, for a flow rate of 10 pL/min, the n value is 7. These values indicate the complexity of the flow pattern and the extent of mixing required in the microchannel design to achieve efficient mixing under these conditions.

Several experiments were performed that analyse the changes in each of these parameters and their effects on mixing efficiency and thus the contacting between the bacteria/pathogen and beads. These results are shown in Figure 4. Figure 4 shows plots of the mixing efficiency against the total liquid flow rate at different pressure in microfluidic chip of different designs shown in the respective insets (a) Design 1 (DI); (b) Design 2 (D2); (c) Design 3 (D3); and (d) Comparing three different designs at optimum pressure with different flowrate. The original concentration of bacteria spiked in blood was IxlO⁴ CFU/mL and the concentration of the beads in the buffer was 4xl0⁹ beads/mL for all experiments conducted. The results in design 3 shows consistently higher mixing efficiencies (Figure. 4d) at all liquid flow rates tested and 20 psi gas pressures.

Example 6

Gas Separation Studies

A novel approach was devised to enhance mixing and further magnetization followed by gas separation and calculate the efficiency of pathogen isolation using a microfluidic chip. This approach involved modifying the channel geometry, specifically implementing Design 3 from the above mixing studies. The three different layers that are aligned parallelly and bonded to form the integrated microfluidic chip device to facilitate gas separation and isolate pathogen from whole blood. The mixing zone containing the D3 design forms layer 2 with 3 wells (Wl, W2, W3) while the magnetization zone containing three wells (W4, W5, W6) to separate the gas and the beads-pathogen complex from the segmented microflow unit forms layer 3 which is the critical steps in the process of the present invention. The separated gas is allowed to escape through the filters placed on layer 1 (as shown in Figure 5).

The process begins by introducing the blood sample containing pathogen via inlet 1, N2 gas via inlet 2, and protein coated magnetic beads with buffer via inlet 3 into the microfluidic chip device through the three inlets on the intermediate layer (layer 2). Simultaneously, the EZ (EZFlow® PES Membrane Disc Filters with pore diameter of 0.45 pm) flow membrane in the upper layer (layer 1) eliminates gas, ensuring that only the desired components progress through the system. The layer 2 (as shown in the Figure 6), where the wells for pathogen capture are located, aligns with the lower layer (layer 3) to facilitate efficient pathogen capture. Once inside the microfluidic chip, the pathogen is captured and removed along with the gas using these wells. Magnets are placed beneath the wells of the layer 3 which assist in attracting the pathogen that are attached to the magnetic beads, ensuring effective separation. The layer 3 of the microfluidic chip features an outlet (outlet 2) for collecting the pathogen bound to the magnetic beads. During the process, the solution is collected in tubes at the outlet 1 of the channel (layer 2), typically around 0.5 mF for the optimized flow rate used in the mixing studies. External magnets are used to attract the magnetic beads downwards, aiding in the collection process. The pathogens are then gathered from the supernatant blood and attached to the beads, all while the magnets are kept activated (as shown in the Figure 7).

Subsequently, the waste blood is collected from the outlet 1 of the layer 2 of the microfluidic chip, with the layer 3 outlet closed to prevent contamination. After collecting the waste blood, the outlet is closed, and PBS buffer is passed through one of the inlets of the chip. The magnets are then removed to collect the pathogen attached to the magnetic beads at the outlet 2 (layer 3) as shown in the Figure 8. These collected pathogen are then carefully spread on agar plates and incubated overnight at 37 °C.

This step is crucial as it allows to assess the concentrations and separation efficiency of the pathogen, providing valuable insights into the effectiveness of the microfluidic chip design.

Following the pathogen isolation process, the isolated pathogens are passed through the TSAW-based micro piezo-actuator (Indian Patent 202341040780 filed on 15^th June 2023). This step helps to lyse the pathogen, breaking down their cellular structures to release the nucleic acids. The nucleic acids contain genetic information unique to each pathogen, which is essential for further analysis and identification as shown in the Figure 9. For specific pathogen like E. coli and S. aureus, specific primer and probe sets are designed. These primer and probe set sequences of nucleotides that are complementary to the target DNA sequences of the pathogen. During the polymerase chain reaction (PCR) process, these primers bind to the target DNA, and the probe binds to the amplified DNA, emitting a signal that indicates the presence of the pathogen.

The PCR process involves multiple cycles of heating and cooling, which help to amplify the target DNA sequences. The amplified DNA is then analysed using endpoint detection method. This allows for the detection and quantification of the pathogen present in the whole blood sample, providing valuable information for diagnosis and further research.

Example 7

The microfluidic chip device

The microfluidic chip device consists of three layers: an upper layer (layer 1), and intermediate layer (layer 2) and a lower layer (layer 3). The intermediate layer (layer 2) has three inlets: inlet 1 for sample containing pathogen, inlet 2 for N2 gas, and inlet 3 for magnetic beads with buffer and one outlet (outlet 1). The intermediate layer (layer 2) also has multiple microfluidic channel and three wells (Wl, W2, W3). The lower layer (layer 3) features three wells (W4, W5, W6) for pathogen capture, which are aligned with the wells (Wl, W2, W3) in the intermediate layer (layer 2). The upper layer (1) is equipped with an EZ flow membrane filter paper (0.45 pm pore diameter) to eliminate gas and contamination. The lower layer (layer 3) also features an outlet (outlet 2) for gathering the pathogen bound to the magnetic beads with buffer.

The device is comprised of two main zones - the mixing and binding zone, and the magnetizing zone. These areas are designed to optimize the interaction between the magnetic beads and pathogen, ensuring effective pathogen capture. Magnetization zone: neodymium magnets (N52) placed beneath the wells (w4, w5, w6) of the layer 3 assist in attracting the pathogen that are attached to the magnetic beads, ensuring effective separation. Wells are marked in the Figure 5. Pathogen are captured and then removed along with gas using wells in the lower layer (layer 3) of the microfluidic chip. Using magnets beneath the wells helps attract the pathogen that are attached to the magnetic beads.

Utilizing Specialized Magnetic Beads: The process involves the use of specialized magnetic beads containing proteins (ApoH-CaptoBAC kit, France) to selectively bind to pathogen present in the blood sample. This specialized coating enhances the accuracy of attaching to the pathogen, thus improving the overall effectiveness of the isolation procedure.

Segmented Microfluidic in the device enables rapid mixing and interaction between magnetic beads and pathogen, leading to a decrease in isolation time and steps, as well as an improvement in separation efficiency.

Pathogen Lysis involves lysing the captured pathogen using a TS AW -based micro-piezo actuator. This allows for rapid pathogen breakdown within seconds while preserving the necessary intracellular components for PCR-based pathogen detection. After lysis of pathogen, PCR is used for detecting the pathogen by amplifying specific DNA sequences.

It should be noted that the description and figures merely illustrate the principles of the present subject matter. It should be appreciated by those skilled in the art that conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present subject matter. It should also be appreciated by those skilled in the art that by devising various systems that, although not explicitly described or shown herein, embody the principles of the present subject matter and are included within its spirit and scope. Furthermore, all examples recited herein are principally intended expressly to be for pedagogical purposes to aid the reader in understanding the principles of the present subject matter and the concepts contributed by the inventor(s) to further the art and are to be construed as being without limitation to such specifically recited examples and conditions. The novel features which are believed to be characteristic of the present subject matter, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. Although embodiments for the present subject matter have been described in language specific to package features, it is to be understood that the present subject matter is not necessarily limited to the specific features described. Rather, the specific features and methods are disclosed as embodiments for the present subject matter. Numerous modifications and adaptations of the system/device of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the scope of the present subject matter.

It will be further appreciated that functions or structures of a plurality of components or steps may be combined into a single component or step, or the functions or structures of one-step or component may be split among plural steps or components. The present invention contemplates all of these combinations. Unless stated otherwise, dimensions and geometries of the various structures depicted herein are not intended to be restrictive of the invention, and other dimensions or geometries are possible. In addition, while a feature of the present invention may have been described in the context of only one of the illustrated embodiments, such feature may be combined with one or more other features of other embodiments, for any given application. It will also be appreciated from the above that the fabrication of the unique structures herein and the operation thereof also constitute methods in accordance with the present invention. The present invention also encompasses intermediate and end products resulting from the practice of the methods herein.

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Claims

CLAIMS:

1. A microfluidic chip device comprising:

(a) an upper layer (layer 1);

2. The microfluidic chip device as claimed in claim 1, wherein an upper layer (layer 1) is equipped with a membrane filter paper to eliminate gas and contamination.

3. The microfluidic chip device as claimed in claim 1, wherein the inlet 1 is for introducing a sample, the inlet 2 is for introducing a gas, the inlet 3 is for introducing magnetic beads with buffer, and outlet 1 is for collection of waste sample.

4. The microfluidic chip device as claimed in claim 1, wherein the outlet 2 is for collecting pathogen bound to the magnetic beads with buffer.

5. The microfluidic chip device as claimed in claim 1, wherein the plurality of wells (Wl, W2, W3) are for capture of pathogen and the plurality of wells (W4, W5, W6) in lower layer (layer 3) are aligned with the plurality of wells (Wl, W2, W3) in intermediate layer (layer 2).

6. The microfluidic chip device as claimed in claim 1, wherein the microfluidic channels are squiggle in shape and have dimension of 300 pm.

7. The microfluidic chip device as claimed in claim 1, wherein the device is fabricated using a substance selected from the group consisting of poly dimethyl siloxane, silicon, quartz, glass, poly methyl methacrylate, polycarbonate, and cycloolefin polymer.

8. A method for isolating a pathogen from a body fluid sample comprising:

(ii) maintaining the sample flow rate in the range of 1 pL/min to 10 pL/min and gas pressure in the range of 20 psi and 30 psi in microfluidic channels; (iii) allowing capture of pathogen attached to the protein coated magnetic beads in the plurality of wells (Wl, W2, W3);

(iv) placing a magnet beneath the plurality of wells (W4, W5, W6) to attract pathogen attached to the magnetic beads;

(v) collecting pathogen attached to the magnetic beads from outlet 2;

(vi) separating the pathogen from the magnetic beads; and

(vii) collecting waste sample from outlet 1.

9. The method as claimed in claim 8, wherein the method further comprises passing the separated pathogen through a micro piezo-actuator for lysis to release nucleic acid.

10. The method as claimed in claim 9, wherein the method further comprises conducting an assay on the nucleic acid to identify the pathogen.

11. The method as claimed in claim 10, wherein the assay is selected from the group consisting of a sequencing reaction and a polymerase chain reaction.

12. The method as claimed in claim 8, wherein the body fluid sample is selected from the group consisting of serum, blood, sweat, semen, cerebrospinal fluid, saliva, tears, urine, vaginal secretions, synovial fluid, pleural fluid, pericardial fluid, nasal fluid, gastric fluid, and breast milk.

13. The method as claimed in claim 8, wherein the pathogen is selected from the group consisting of fungi, bacteria, virus or a combination thereof.