Claims:1.A portable device for detecting an analyte, the device comprising:
a microfluidic chip comprising at least three microfluidic channels, a first of said at least three microfluidic channels being configured to receive a metal precursor at its inlet, a second of said at least three microfluidic channels being configured to receive an analyte containing sample at its inlet, and a third of said at least three microfluidic channels being configured to receive a reducing agent at its inlet; wherein the at least three microfluidic channels connect to at least one reaction channel that is configured with a micro-mixing geometry to allow reaction between the metal precursor and the reducing agent to form metal nanoparticles, wherein presence of the analyte interrupts synthesis of the metal nanoparticles;

and at least one optical detection system configured to measure changes in the optical absorbance or localized surface plasmon resonance of the metal nanoparticles whose formation/size/shape/aggregates gets modified/influenced as a result of interruption/interference of the synthesis process in proportion to the concentration of the analyte present in the analyte containing sample, wherein the measured change in the optical absorbance or localized surface plasmon resonance is used for a quantitative assessment of the analyte in the at least one sample.

2. The device of claim 1, wherein the device further comprises a pumping system configured to induce flow of the at least one metal precursor, the at least one analyte containing sample, and the at least one reducing agent through microfluidic chip.

3. The device of claim 2, wherein the pumping system is a vacuum based pumping system.

4. The device of claim 3, wherein the at least one reaction channel is connected to an outlet channel and the vacuum based pumping system is configured to the outlet channel to induce suction of the at least one metal precursor, the at least one analyte containing sample, and the at least one reducing agent through the inlets of the respective microfluidic channels, and wherein the vacuum based pumping system further induces their flow through the reaction channel, and wherein the microfluidic channels and the reaction channel are dimensioned to control flow of the precursor, the analyte containing solution, and the reducing agent to their respective desired values.

5. The device of claim 3, wherein the vacuum based pumping system is a syringe pump.

6. The device of claim 1, wherein the reducing agent is a plant extract, wherein the plant extract is obtained by boiling leaves or any other parts of Partheniumhisterophorus.

7. The device of claim 1, wherein the at least one optical detection system incorporates
a light emitting module configured to provide an incident light for irradiating the synthesised metal nanoparticles, and
a light detecting module configured to measure from emergent light changes in absorbance or localized surface plasmon resonance peak of the synthesised metal nanoparticles.

8. The device of claim 1, wherein the at least one reaction channel is serpentine in nature with length in the range of 5 to 60 cm and width in the range of 10 to 1000 µm.

9. The device of claim 1, wherein width of the at least three microfluidic channels is in the range of 10 µm to 500 µm.

10. The device of claim 1, wherein the microfluidic chip is made of PDMS or Polymethyl methacrylate (PMMA) or glass.

11. The device of claim 1, wherein the at least three microfluidic channels further comprise a reference channel being configured to receive a reference sample at its inlet and the reference channel along with the first channel and the third channel connects to the at least one reaction channel that is configured with a micro-mixing geometry to allow reaction between the metal precursor and the reducing agent to form metal nanoparticles in presence of the reference sample.

12. A method for detecting an analyte comprising steps of:

allowing a microfluidic chip to receive at least one metal precursor, at least one reducing agent, and at least one analyte containing sample;
actuating a pumping system that is operatively coupled with the microfluidic chip to enable the at least one metal precursor, the at least one reducing agent, and the at least one analyte containing sample to flow to at least one micromixing channel to allow reaction between the at least one metal precursor and the at least one reducing agent for interruption-synthesis of metal nanoparticles in presence of the analyte present in the at least one analyte containing sample; and
measuring localized surface plasmon resonance of the synthesized metal nanoparticles to make a quantitative assessment of the analyte in the at least one sample.

13. The method of claim12, wherein the at least one reducing agent is a plant extract and the plant extract is obtained by boiling leaves or any other parts of Partheniumhisterophorus.

14. The method of claim 12, wherein the at least one reducing agent is a chemical reducing agent selected from group consisting of PVP, sodium borohydride.

15. The method of claim 12, wherein the metal precursor is a salt form of a metal selected from a group comprising of gold, silver, cobalt, copper, platinum and palladium.

16. The method of claim 15, wherein the salt is selected from a group comprising of sulphates, silicates, nitrates, nitrides, oxides, sulfides and chlorides.

17. The method of claim 12, wherein the reaction between the at least one metal precursor and the at least one reducing agent for interruption synthesis of the metal nanoparticles in the presence of the analyte present in the at least one analyte containing sample takes place at room temperature.

18. The method of claim 12, wherein the measurement of localized surface plasmon resonance of synthesized metal nanoparticles is done using a optical detection system that incorporates a light emitting module configured to provide an incident light for irradiating the synthesised metal nanoparticles and a light detecting module configured to measure from emergent light, changes in localized surface plasmon resonance of the synthesised metal nanoparticles.

19. The method of claim 12, wherein flow rate of the at least one metal precursor and the at least one analyte containing sample is in the range of from 50µL/min to 2000 µl/min; and the flow rate of the reducing agent is in the range of from 0.1 µL/min to 50 µl/min.

20. The method of claim 12, wherein the analyte is melamine and the analyte containing sample is milk.
, Description:FIELD OF THE INVENTION
[0001] The present disclosure pertains to technical field of analyte detection. In particular, the present disclosure relates to a microfluidic sensor device, and a method for real time preprocessing of raw samples containing a target analyte and detection of the target analyte by on-chip interference-synthesis of noble metal nanoparticles using such device.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Noble metal nanoparticles are being widely used for sensing applications due to their plasmonic properties and good extinction coefficient. Surface Plasmon Resonance (SPR) of a metal nanoparticle is mainly related to nanoparticle size, shape, composition, inter-particle distance and dielectric constant (refractive index) of surrounding medium. Among the metal nanoparticles known to exhibit SPR, silver nanoparticles have an especially strong SPR and are expected to provide particularly high sensitivity for sensing purposes.
[0004] Several microfluidic devices have been developed in the art utilizing the plasmonic properties of metal nanoparticles that allow qualitative or quantitative detection of target "analytes" also called target "molecules", such as food adulterants (e.g. melamine), ammonia, DNA, heavy metal ions, explosives, pollutants, pesticides, etc.
[0005] Tong et al. (Optical aggregation of metal nanoparticles in a microfluidic channel for surface-enhanced Raman scattering analysis. Lab on a Chip, 9(2), 193-19) describes a microfluidic device based sensing of environmental pollutants by gold nanoparticles. In this method, the color change which may be observed in presence of a target analyte is caused primarily by aggregation of nanoparticles rather than a change in size of the nanoparticles leading to change in SPR.The individual gold nanoparticles are synthesized off-chip and functionalized such that the analyte causes them to aggregate, giving rise to a color change.
[0006] Devices for detection of target analytes using nanoparticles synthesis-interruption in presence of the analytes have also been reported in the art (Yin et al. Rapid colorimetric detection of melamine by H2O2–Au nanoparticles. RSC Advances, 5(42), 32897-32901;and Zhang et al. Colorimetric detection of melamine based on the interruption of the synthesis of gold nanoparticles. Analytical Methods, 5(8), 1930-1934). However, these devices and methods rely upon off-chip synthesis of metal nanoparticles which method is generally time consuming and thereby making real-time detection of target analytes virtually impossible.
[0007] Further, all these known methods, produce metal nanoparticles chemically, i.e. the known methods require presence of strong synthetic reducing agents which are carcinogenic and hazardous to the environment, and may also require high process temperature and stirring using magnetic bar or shaking at some point of reaction step thereby making the process energy inefficient and expensive. Further, the applicability of known sensors for detection of analytes is limited due to their low sensitivity and the requirement of bulk volume of test sample.
[0008] There is thus a need in the art for a device that is capable of quantitative and qualitative detection of target analytes having the advantages of real time detection of target analytes, high selectiveness, easy to operate, requiring less sample volume, energy efficient, environment friendly and quick reaction speed. Also, there is a need in the art for real time detection of target analytes using interference effects by the analytes in biosynthesis of noble metal nanoparticles using such microfluidic device.
[0009] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION
[00010] It is an object of the present disclosure to provide a portable device capable of being used for real time detection of a target analyte in test samples.
[00011] It is a further object of the present disclosure to provide a portable device that facilitates real time detection of the target analyte through interference of the analyte in synthesis of noble metal nanoparticles.
[00012] It is another object of the present disclosure to provide a portable device that facilitates real time detection of target analytes based on microfluidic on-chip interference-synthesis (IS) of metal nanoparticles using plant extract as reducing agent. It is another object of the present disclosure to provide a portable device that can facilitate real time detection of target analytes at ambient temperature.
[00013] It is another object of the present disclosure to provide a portable device that can facilitate accurate detection of target analytes with relatively lower sample volumes.
[00014] It is another object of the present disclosure to provide a portable device that is simple in structure, easy to operate and is highly energy efficient.
[00015] It is another object of the present disclosure to provide a simple and rapid method for detection of target analytes in test samples.
[00016] It is another object of the present disclosure to provide a colorimetric method for detection of target analytes based on interference effects in microfluidic on-chip synthesis of metal nanoparticles.
[00017] It is another object of the present disclosure to provide a device having a preprocessing unit for processing of raw samples containing the analyte using a Microfluidic reactor for subsequent detection of the same.

SUMMARY OF THE INVENTION
[00018] Aspects of the present disclosure relate to a portable device and method for real time detection of a target analyte through microfluidic on-chip interference-synthesis (referred to as IS which signifies the analyte interrupting / interfering the synthesis of nanoparticles as against SS which signifies as synthesized nanoparticles being used subsequently for sensing of an analyte) of noble metal nanoparticles. In an aspect, the proposed method is based on measurement of optical absorbance and/or localized surface plasmon resonance due to formation/size/shape/aggregates of the synthesized nanoparticles which gets modified/influenced as a result of interruption/interference of the synthesis process in proportion to the concentration of the analyte during the synthesis process. In an aspect, the disclosed method can enable real time detection of target analytes based on microfluidic on-chip interference synthesis of metal nanoparticles using plant extract as reducing agent.
[00019] In an embodiment, the device can include (a) a microfluidic chip incorporating at least three microfluidic channels, at least one of the at least three microfluidic channels configured to receive a metal precursor at its inlet, at least one of the at least three microfluidic channels configured to receive an analyte containing solution at its inlet and at least one of the at least three microfluidic channels configured to receive a reducing agent at its inlet; the at least three microfluidic channels joining together to form at least one reaction channel configured with a micro-mixing geometry to allow reaction between the metal precursor, analyte and the reducing agent to form metal nanoparticles at room temperature; further the at least one reaction channel connected to an outlet channel; (b) an optical detection system to measure changes in localized surface plasmon resonance of metal nanoparticles, the detection system incorporating a light emitting module configured to provide an incident light for irradiating the metal nanoparticles; and a light detecting module configured to measure from emergent light, changes in localized surface plasmon resonance of metal nanoparticles.
[00020] In an embodiment, the device can further incorporate a pumping system to facilitate flow of fluids through the plurality of microfluidic channels configured on the microfluidic chip wherein the pumping system can for example be a vacuum based pumping system configured to the outlet channel and can among various option comprise a syringe that can be pulled and locked in position to create suction to flow the fluids through the plurality of microfluidic channels and the reaction channel.
[00021] According to embodiments of the present disclosure, the microfluidic chip can be formed of polydimethylsiloxane or glass. The length of the reaction channel can preferably be in the range of from 1 mm to 50 mm, and the width can preferably range from 10 µm to 1000 µm. The width of the microfluidic channels and the outlet channel can preferably be in the range of from 50 µm to 400 µm.
[00022] In another aspect, the present disclosure provides a real time on-chip interruption-synthesis of nanoparticles based method for detecting an analyte using a plant extract as reducing agent, wherein the method can include steps of (a) providing a microfluidic chip configured to receive at least one metal precursor, at least one reducing agent and at least one analyte containing sample and direct them to at least one micromixing channel to allow reaction between the metal precursor, analyte and the reducing agent to form metal nanoparticles; (b) providing a pumping system operatively coupled to the microfluidic chip and configured to induce flow of the at least one metal precursor, the at least one analyte containing sample and the at least one reducing agent through the microfluidic chip wherein channels in the microfluidic chip are dimensioned to control flow of the at least one precursor, the at least one analyte containing sample and the at least one reducing agent at their respective desired values wherein the pumping system can be a vacuum based pumping system that can induce suction and subsequent flow of the at least one metal precursor, at least one reducing agent and at least one analyte containing sample and direct them to at least one micro-mixing section of the chip; (c) allowing the microfluidic chip to receive the at least one metal precursor, the at least one reducing agent and the at least one analyte containing sample; (d) actuating the pumping system to enable the at least one metal precursor, the at least one reducing agent and the at least one analyte containing sample to flow to the at least one micromixing channel to allow reaction between the at least one metal precursor and the at least one reducing agent for interruption-synthesis of metal nanoparticles in presence of the analyte present in the analyte containing sample; and (e) measuring localized surface plasmon resonance of synthesized metal nanoparticles to make a quantitative assessment of the analyte in the at least one sample wherein the measurement of localized surface plasmon resonance of synthesized metal nanoparticles can be done using a optical detection system that incorporates a light emitting module configured to provide an incident light for irradiating the synthesised metal nanoparticles and a light detecting module configured to measure from emergent light, changes in localized surface plasmon resonance of the synthesised metal nanoparticles. In an aspect, the at least one reducing agent can be a plant extract such as extract obtained by boiling leaves of Partheniumhisterophorus or Lawsoniainermis.
[00023] In one embodiment, the metal precursor can be a salt form of a metal selected from the group consisting of gold, silver, cobalt, copper, platinum and palladium. The salt form can be sulphates, silicates, nitrates, nitrides, oxides, sulfides or chlorides.
[00024] In another embodiment, the reducing agent used in the synthesis of metal nanoparticles can be a plant extract and can be prepared by boiling leaves of a plant in water to extract water soluble components.
[00025] In yet another embodiment, the plant extract can be prepared by boiling leaves of Partheniumhisterophorus in water.
[00026] In an embodiment, the disclosed device can be used to detect food adulterants, such as melamine. The disclosed device can in particular be used for detecting adulteration of milk by melamine containing adulterants that make the milk look protein rich due to high Nitrogen content of melamine but may lead to formation of kidney stones in adults and infants.
[00027] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS
[00028] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[00029] FIG. 1 illustrates an exemplary schematic arrangement for a portable device for detection of an analyte through microfluidic on-chip interference synthesis of metal nanoparticles in accordance with embodiments of the present disclosure.
[00030] FIG. 2 illustrates an exemplary working model of a portable device for detection of an analyte through microfluidic on-chip interference synthesis of metal nanoparticles in accordance with embodiments of the present disclosure.
[00031] FIG. 3A illustrates a preferred configuration of a microfluidic chip for on-chip interference synthesis of metal nanoparticles in accordance with embodiments of the present disclosure.
[00032] FIG. 3B illustrates another exemplary configuration of a microfluidic chip for on-chip interference synthesis of metal nanoparticles configured to simultaneously checking a reference sample and a test sample in accordance with embodiments of the present disclosure.
[00033] FIG. 4A is an exemplary image showing on-chip sensing of melamine at 0.1 ppm, 1 ppm, 10 ppm, 100 ppm, 1000 ppm concentration, using Partheniumhisterophorus leaf extract as reducing agent in the interference biosynthesis of silver nanoparticles, in accordance with embodiments of the present disclosure.
[00034] FIG. 4B is an UV-VIS spectroscopy exhibiting spectral shift in LSPR peak of silver nanoparticles synthesized using the microfluidic chip with different ppm concentration of melamine, in accordance with embodiments of the present disclosure.
[00035] FIG. 5A is included for comparative purpose and illustrates an off-chip sensing of melamine (0.1 ppm, 1 ppm, 10 ppm, 100 ppm, 1000 ppm concentration) using Partheniumhisterophorus leaf extract as reducing agent in the interference biosynthesis of silver nanoparticles, in accordance with embodiments of the present disclosure.
[00036] FIG. 5B is included for comparative purpose and illustrates a UV-VIS spectroscopy graph exhibiting spectral shift in LSPR peak of silver nanoparticles synthesized off-chip with different ppm concentration of melamine.
[00037] FIG. 6 illustrates different scales of High Resolution Transmission Electron Microscopy images of silver nanoparticles biosynthesized on-chip using the microfluidic chip with (100ppm) and without melamine in accordance with embodiments of the present disclosure.
[00038] FIG. 7 is included for comparative purpose and illustrates High Resolution Transmission Electron Microscopy images of silver nanoparticles biosynthesized off-chip with (100ppm) and without melamine in accordance with embodiments of the present disclosure.
[00039] FIGs. 8A and 8B are three dimensional graphs illustrating a comparison between SS and IS sensing of melamine of 1ppm and 10ppm concentration respectively, in accordance with embodiments of the present disclosure.
[00040] FIG. 9A illustrates a UV-VIS absorption graph exhibiting off-chip biosynthesized silver nanoparticles used for detection of melamine at different hours, in accordance with embodiments of the present disclosure.
[00041] FIG. 9B illustrates a UV-VIS absorption graph exhibiting on-chip biosynthesized silver nanoparticles used for off-chip detection of melamine, in accordance with embodiments of the present disclosure.
[00042] FIGs. 10A and 10B illustrate time kinetics comparison of off-chip and on-chip interference biosynthesis of silver nanoparticles with (1 ppm) and without melamine for colorimetric detection of melamine, in a 2 dimension and 3 dimension configuration respectively.
[00043] FIG. 11A depicts an exemplary experimental setup of the present device that includes a laser diode light source of 405nm wavelength and OPT101 photo detector for detection of melamine in accordance with embodiments of the present disclosure.
[00044] FIG. 11B is a sensing graph illustrating different ppm concentration of melamine after detection in accordance with embodiments of the present disclosure.
[00045] FIG. 12A illustrates results of selectivity test carried out using different analytes at 100 ppm concentration through interference synthesis of silver nanoparticles in accordance with embodiments of the present disclosure.
[00046] FIG. 12B is an exemplary graph showing, among other analytes, least absorbance of melamine at 416 nm in accordance with embodiments of the present disclosure.
[00047] FIG. 13 illustrates mechanism of melamine interruption in the biosynthesis of silver nanoparticles using Partheniumhisterophorus leaf extract as reducing agent in accordance with embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[00048] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered 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 as defined by the appended claims.
[00049] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[00050] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[00051] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[00052] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
[00053] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[00054] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[00055] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[00056] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[00057] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[00058] Embodiments of the present disclosure relate to a device and method for real time detection of target analyte through on-chip interference-synthesis of metal nanoparticles.In an aspect, the proposed method is based on measurement of optical absorbance and/or localized surface plasmon resonance due to formation/size/shape/aggregates of the synthesized nano particles which gets modified/influenced as a result of interruption/interference of the synthesis process in proportionate to the concentration of the analyte during the synthesis process.
[00059] In an aspect, the disclosed method can facilitate real time detection of target analytes based on microfluidic on-chip interference synthesis of metal nanoparticles using plant extract as reducing agent. The disclosed device can have precise control over growth and size distribution of resulting metal nanoparticles, and thereby enables effective and accurate detection of target analyte at room temperature.
[00060] It is to be understood that though various embodiments of the present disclosure have been explained with reference to detection of melamine- an adulterant commonly used in milk, these can be suitably modified by a person skilled in art to detect and measure various other analytes/contaminants/pollutants/adulterants in fluid samples and all such applications and modifications are well within the scope of the present disclosure.
[00061] In an embodiment, the disclosed device is based on a microfluidic chip and further comprises a pumping system that facilitates flow of required fluids through microfluidic channels configured on the microfluidic chip.
[00062] In an embodiment, the microfluidic channels of the microfluidic chip can be configured to control the flow of the required fluids through the channels at their respective desired values to facilitate precise control over growth and size distribution of metal nanoparticles synthesized during the process, and thereby enabling effective detection of target analyte through changes in surface plasmon resonance (SPR) of resulting metal nanoparticles on account of analyte interruption in the synthesis of metal nanoparticles.
[00063] In an embodiment, the disclosed device can incorporate an optical detection system comprising a light emitting module that can output a light of specific wavelength to irradiate the metal nanoparticles synthesized during the process and a light detecting module employed to measure from the emergent light, changes in optical absorbance and/or surface plasmon resonance of resulting metal nanoparticles. The change in optical absorbance and/or surface plasmon resonance of the metal nanoparticles can be due to difference in metal nanoparticle morphologies on account of nanoparticle synthesis interruption caused by presence of the analyte, and it can be measured for quantitative and qualitative detection of the analyte.
[00064] As used herein, the term "analyte" refers to a molecule present in a test sample whose determination is of interest to a user. Representative examples of analytes can include food adulterants (e.g. melamine), pesticides, herbicides, ammonia, heavy metal ions, environmental pollutants, hazardous chemicals, explosives and the like.
[00065] In an embodiment, the present disclosure provides a method for real time detection of target analytes through microfluidic on-chip interference biosynthesis of metal nanoparticles. According to embodiments, the metal nanoparticles can be biosynthesized using a metal precursor and a plant extract as reducing agent in presence of an analyte to be detected. The metal precursor can be a salt form of a metal selected from the group consisting of gold, silver, cobalt, copper, platinum and palladium. The salt form can be sulphates, silicates, nitrates, nitrides, oxides, sulfides or chlorides.
[00066] In another embodiment, the plant extract used as reducing agent in the interference synthesis of metal nanoparticles can be prepared by boiling leaves of a plant in water to extract water soluble components. In an aspect use of a plant extract as a reducing agent can prevent use of chemicals that are carcinogenic and hazardous to the environment.
[00067] In yet another embodiment, the plant extract can be prepared by boiling leaves or other parts of Partheniumhisterophorus in water.
[00068] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.
[00069] Referring to FIG. 1 that discloses an exemplary schematic arrangement for a portable device 100 for colorimetric detection of target analyte through microfluidic on-chip interference of biosynthesis of metal nanoparticles in accordance with embodiments of the present disclosure. The device 100 can include a microfluidic reactor 102 (also referred as microfluidic chip or 102 simply as chip 102 hereinafter), a pumping system 104 that can be for example a vacuum based pumping system such as a syringe which can be pulled to create a vacuum and locked in position to facilitate suction and flow of metal precursor, reducing agent and analyte containing sample through the channels of the microfluidic chip 102.Though the exemplary embodiment of the device is configured to carry out colorimetric detection of target analyte through microfluidic on-chip interference of biosynthesis of metal nanoparticles at room temperature, it will be evident to those skilled in the art that the microfluidic reactor 102 can also incorporate microheaters or microcoolers for interference synthesis of nanoparticle at an elevated temperature if the situation or application demands.
[00070] The device can further incorporate reservoirs such as a reducing agent reservoir 110, a precursor reservoir 108 and an analyte containing sample reservoir 106, wherein these reservoirs can be configured at inlets of the channels of the microfluidic chip 102 to help suction of the leaf extract, the metal precursor and the sample under influence of the vacuum generated by the vacuum based pumping system. There can be digitally controlled microfluidic valves configured between the respective reservoirs and the corresponding inlets of the channels of the microfluidic chip 102 to control flow of the reducing agent, the metal precursor and the sample to the chip 102.
[00071] In an embodiment, the device 100 can further incorporate a pre-processing unit 118 configured to pre-process analyte containing raw samples (such as milk) to extract / separate the analyte before subjecting it to sensing by interference sensing. In an aspect the preprocessing unit 118 may be located in the microfluidic reactor 102 itself or it can be a separate microfluidic device.
[00072] In an embodiment, the device 100 can further incorporate an optical detection system that may comprise a light emitting module 112 that can output a light of specific wavelength to irradiate the metal nanoparticles synthesized during the process, and a light detecting module 114 to measure from the emergent light changes in localized surface plasmon resonance (LSPR) of the synthesized metal nanoparticles.
[00073] In an embodiment, the device 100 can incorporate a microfluidic detection zone 116 configured to receive the synthesized nano particles where they can be subjected to measurement for change in localized surface plasmon resonance using the optical detection system comprising the light emitting module 112 and the light detecting module 114. In an aspect the a microfluidic detection zone 116 can be a microfluidic collection device configured between the outlet of the microfluidic chip 102 and the pumping system 104.
[00074] In an embodiment, the device 100 can further comprise a micro controller 120 operatively coupled to the digitally controlled microfluidic valves, the light emitting module 112 and the light detecting module 114 to control their operation and interpret the emergent light changes detected by the light detecting module 114 for qualitative and quantitative assessment of presence of the target analyte in the sample. The device can additionally incorporate a display 122 operatively coupled to the micro controller 120 to display the test results.
[00075] FIG. 2 illustrates an exemplary working model of the device for colorimetric detection of target analyte through microfluidic on-chip interference biosynthesis of metal nanoparticles in accordance with embodiments of the present disclosure. The light emitting module 112 can provide an incident light for irradiating the metal nanoparticles synthesized on-chip in presence of an analyte. The light emitting module can be configured to provide a monochromatic light, polychromatic light, a narrow-band light, or a white light. Preferably, a laser diode outputting a monochromatic light having a wavelength corresponding to surface plasmon resonance wavelength of the resulting metal nanoparticles can be used. The light detecting module 114 can detect an emergent light so as to measure from the emergent light changes in aoptical absorbance and/or localized plasmon resonance of resulting metal nanoparticles. An Ultra Violet spectrometer may be used as a light detection module to detect a wavelength of light emitted from the surfaces of the metal nanoparticles synthesized during the process. The light detection module 114 can detect the surface plasmon resonance wavelength changed due to change in nanoparticle morphologies on account of analyte interruption in the biosynthesis process. Also, the light detecting module 114 can be configured to analyze the change of optical absorbance and/or localized surface plasmon resonance wavelength so as to quantify the concentration of analyte to be analyzed.
[00076] FIG. 3 illustrates a preferred configuration 300 of the microfluidic chip 102 for on-chip interference synthesis of metal nanoparticles using plant extract as reducing agent in accordance with embodiments of the present disclosure. The microfluidic chip 102 can comprise a first channel 302 having a first inlet 308, a second channel 304 having a second inlet 310 and a third channel 306 having a third inlet 312. The first, second and third channels can lead to a reaction section 314 in which chemical reaction between the reactants i.e. the metal precursor and the reducing agent, can take place in presence of the analyte to be detected in the sample. The reaction section 314 can include at least one reaction channel 316 that can provide aging length for the growing nanoparticles. The reaction channel 316 can be provided with at least one micro-mixing geometry318 that can have a specific flow design throughout the flow path to enhance mixing of the reactants. The microfluidic chip 102 can include at least one outlet or exit channel 320through which the synthesized nanoparticles may exit the chip 102. The outlet channel 320 can be fluidly coupled to the reaction channel 316 and can be positioned downstream from the reaction section 314.
[00077] The microfluidic chip 102 can be formed of glass or an optically transparent polymeric material such as Polymethyl methacrylate (PMMA) or PDMS and can be fabricated using any method known in relevant art. In a preferred embodiment, the microfluidic chip 102 can be fabricated through lithography.
[00078] In an embodiment, the first channel 302, second channel 304 and the third channel 306 can be dimensioned to control the flow of fluids under the suction of the vacuum based pumping system at their respective desired flow rates wherein the desired flow rate can depend on the type of analyte to be detected through interference synthesis and choice of corresponding precursor and reducing agent. In an aspect, microfluidic chip can be fabricated to meet the requirement of synthesis of different metal nanoparticles using different combinations of the precursor and the reducing agent in presence of an analyte to be detected, therefore, it is to be appreciated that FIG. 3 is purely exemplary and the features of the microfluidic chip 102, such as, the inlets 308, 310 and 312, outlet 320, reaction channel 316 and the micromixing section 318 can take any other size, length and shape desired to suite the intended purpose and use.
[00079] The reaction channel 316 can be serpentine in nature with effective length in the range of 5to 60 cm and width in the range of 10 to 1000 µm. The width of the first, second and third channels and outlet channel can preferably be in the range of 10 µm to 500 µm.
[00080] In an embodiment, the micro-mixing section 318 can be a thin and long channel in which complete mixing of the metal precursor, plant extract and analyte containing solution can occur in approximately less than one second. The micromixing section 318 can have a specific flow design throughout the flow path to enhance mixing of the reactants and analyte. The micromixing section 318 can have a planar geometry that enables effective mixing of the metal precursor, plant extract and analyte within a very small foot-space and thereby reducing the quantity of analyte sample needed for the detection.
[00081] The reactants such as the metal precursor and reducing agent and the analyte containing solution can be made to flow through the microfluidic channels in the microfluidic chip 102. For example the pumping system can be but limited toa vacuum based pumping system that can induce suction of the reactants such as the metal precursor and reducing agent and the analyte containing solution through the inlets 308, 310 and 312 under influence of vacuum generated by suitable means. It is to be understood that though in the exemplary embodiment a syringe 104 has been used as a vacuum based pumping system, any other means such as a peristaltic pump, an aspirator or a vacuum pump that can made the reactants flow through the chip at desired flow rates, can be used for the purpose without any limitation. The reactants and the analyte can get combined in the reaction channel 316 for effecting chemical reactions which may take place at a very fast rate. The flow rates of the reactants and analyte can be precisely controlled by configuring the microfluidic channels with appropriate dimensions. In an exemplary embodiment, the optimal flow rate of metal precursor and the analyte can be in the range of from 100 µL/min to 2000 µl/min, while the flow rate of plant extract can from 0.1 µL/min to 50 µl/min.
[00082] FIG. 3B illustrates another exemplary configuration 350 of a microfluidic chip 102 for on-chip interference synthesis of metal nanoparticles configured for a comparative interruption synthesis and can simultaneously check a reference sample and a test sample in accordance with embodiments of the present disclosure. The configuration 350 can incorporate a reference arm 352 to receive the reference/control fluid through inlet 356 along with the precursor and the reducing agent through inlets 360 and 362 respectively, and a sample arm 354to receive a test sample (containing analyte/contaminant/pollutant/adulterant to be detected) through inlet 358 along with the precursor and the reducing agent through inlets 360 and 362 respectively. There can be an optical detection zone364 for each of the measurement arm and a reference arm for measurement of absorbance each of the samples. In an aspect, simultaneous measurement on a reference or control sample along with a sample containing the analyte can greatly enhance accuracy, reliability and detection limits of sensing/estimating analyte concentration.
[00083] In an embodiment, the present device 100 can be used for the detection of melamine, a food adulterant through the synthesis of silver nanoparticles in presence of melamine, wherein silver nitrate can be used as the precursor and Partheniumhisterophorus leaf extract can be used as reducing agent. The measurement of optical absorbance and/or localized surface plasmon resonance of the formed silver nanoparticles can allow quantitative detection of melamine.
[00084] In another aspect, the present disclosure provides a real time on chip interruption-synthesis of nanoparticles based method for detecting an analyte using a plant extract as reducing agent, wherein the method can include steps of (a) providing a microfluidic chip configured to receive at least one metal precursor, at least one reducing agent and at least one analyte containing sample and direct them to at least one micromixing channel to allow reaction between the metal precursor, analyte and the reducing agent to form metal nanoparticles; (b) providing a pumping system operatively coupled to the microfluidic chip and configured to induce flow of the at least one metal precursor, the at least one analyte containing sample and the at least one reducing agent through the microfluidic chip wherein channels in the microfluidic chip are dimensioned to control flow of the at least one precursor, the at least one analyte containing sample and the at least one reducing agent at their respective desired values wherein the pumping system can be a vacuum base pumping system that can induce suction and subsequent flow of the at least one metal precursor, at least one reducing agent and at least one analyte containing sample and direct them to at least one micromixing channel of the chip; (c) allowing the microfluidic chip to receive the at least one metal precursor, the at least one reducing agent and the at least one analyte containing sample; (d) actuating the pumping system to enable the at least one metal precursor, the at least one reducing agent and the at least one analyte containing sample to flow to the at least one micromixing channel to allow reaction between the at least one metal precursor and the at least one reducing agent for interruption-synthesis of metal nanoparticles in presence of the analyte present in the analyte containing sample; and (e) measuring optical absorbance and/or localized surface plasmon resonance of synthesized metal nanoparticles to make a quantitative assessment of the analyte in the at least one sample wherein the measurement of optical absorbance and/or localized surface plasmon resonance of synthesized metal nanoparticles can be done using a optical detection system that incorporates a light emitting module configured to provide an incident light for irradiating the synthesised metal nanoparticles and a light detecting module configured to measure from emergent light, changes in optical absorbance and/or localized surface plasmon resonance peak of the synthesised metal nanoparticles.
[00085] In one embodiment, the device used in the disclosed method can be the device 100 and the metal precursor can be dissolved in a suitable solvent to produce a solution which in turn can be placed into the reservoir 108. The metal precursor can be a salt form of a metal selected from the group consisting of gold, silver, cobalt, copper, platinum and palladium. The salt form can be sulphates, silicates, nitrates, nitrides, oxides, sulfides or chlorides.
[00086] In another embodiment, the reducing agent in the synthesis of metal nanoparticles can be placed in the reservoir 110. In an embodiment, the reducing agent can be a plant extract and can be prepared by boiling leaves of a plant in water to extract water soluble components.
[00087] In another embodiment, the analyte containing solution can be placed in reservoir 106. Instead, the analyte can be mixed with metal precursor and the resulting mixture can be placed in the reservoir 108.
[00088] In another embodiment, the disclosed device and method can be used to detect melamine, a food adulterant using leaf extract of Partheniumhisterophorusas reducing agent.

EXAMPLES
[00089] The present disclosure is further explained with help of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
[00090] Detection of melamine based on microfluidic on-chip interference synthesis of silver nanoparticles using the device 100 of the present disclosure: Plant extract was prepared by boiling 30 grams of leaves of Partheniumhisterophorus (a notorious weed) in 100 ml de-ionized water for one hour. The extract was filtered through Whatman Paper and stored at 4oC. The pH of the leaf extract was adjusted to 10 by the addition of 0.1N NaOH.1mM of aqueous silver nitrate solution was received into the inlet 108 along with melamine, and the pH adjusted leaf extract was received in another inlet 110 of the microfluidic chip at different flow rates. Vacuum pump 104 was actuated to flow the silver nitrate solution that contains analyte melamine and the leaf extract through the microfluidic channels and further into the reaction channel at respective desired flow rates. The optimal flow-rates for on-chip biosynthesis of silver nanoparticles were found to be 500 µl/min for metal precursor with different melamine concentrations (0.1 ppm to1000 ppm) and 10µl/min for Parthenium leaf extract. Formation of silver nanoparticles with different ppm concentration of melamine was visually observed by the color change that starts from the junction of the inlets of the microfluidic channels to the outlet 116 of the microfluidic chip. Flow rates were optimized based on the Surface Plasmon Resonance (SPR) band of formed silver nanoparticles.
[00091] Microliter volumes of the formed silver nanoparticles were taken in a 96-well plate and subjected to UV-VIS absorption spectroscopy scan from 300 nm to 800 nm using the TECAN 200 INFINITE plate reader. Silver nanoparticles formed at the optimized flow rates (500:10 µl/min) had a narrow peak at 416 nm. Melamine (0.1 to 1000 ppm concentration) was detected by change in color intensities of silver nanoparticles solution obtained at the outlet 216 and its concentration was determined using UV-VIS spectroscopy. FIG.4A illustrates change in color of the resulting silver nanoparticle solutions which is proportional to concentration of melamine. As shown in FIG. 4B, there was shift in the LSPR peak due to differential size of the formed silver nanoparticles on account of interaction of melamine with phytochemical components of Partheniumhisterophorus leaf extract.
[00092] FIG. 5Aand 5B has been included for comparative purposes, wherein FIG. 5Aillustratesoff-chip sensing of melamine at different ppm concentrations of 0.1 ppm, 1 ppm, 10 ppm, 100 ppm, 1000 ppm, using Partheniumhisterophorus leaf extract biosynthesized silver nanoparticles by means of melamine interference in the synthesis. FIG. 5B depicts a UV-VIS spectroscopy graph exhibiting the spectral shift in LSPR peak of silver nanoparticles synthesized off-chip with different ppm concentration of melamine.
[00093] FIG. 6 illustrates High Resolution Transmission Electron Microscopy (HRTEM) images of "control silver nanoparticles" and silver nanoparticles synthesized on-chip in presence of melamine (100 ppm) at different scales of 2nm, 20nm, 50nm, 100nm. The HRTEM analysis revealed that the difference in size and shape of the resulting nanoparticles was due to interference of melamine in the on-chip biosynthesis of silver nanoparticles. Silver nanoparticles synthesized in absence of melamine (Control silver nanoparticles) exhibited uniform monodisperse nanoparticles of size between 5 to 20nm, while melamine interfered biosynthesis of silver nanoparticles resulted in particles of irregular shape and size. This lead to change in SPR properties and hence there was change in color.
[00094] FIG. 7 has included for comparative purpose that illustrates a High Resolution Transmission Electron Microscopy (HRTEM) images of silver nanoparticles biosynthesized off-chip with and without melamine presence, at different scales of 2nm, 20nm, 50nm, 100nm.
[00095] FIGs. 8A and 8Bare three dimensional graphs illustrating a comparison between SS and IS sensing of melamine of 1ppm and 10ppm concentration respectively. There was no much change between control and SS samples, whereas there was a difference between the silver nanoparticles (synthesized on-chip in presence of melamine) and control samples.
[00096] Off-chip biosynthesized silver nanoparticles were tested with melamine (100 ppm) and it was found that there was not much change in the absorbance for 12 hours. Similarly silver nanoparticles biosynthesized on-chip was tested against different concentration of melamine and no significant absorbance change was observed. FIG. 9A illustrates a UV-VIS absorption graph exhibiting off-chip biosynthesized silver nanoparticles used for detection of melamine at different hours. FIG. 9B illustrates a UV-VIS absorption graph exhibiting on-chip biosynthesized silver nanoparticles used for off-chip detection of melamine. The reason was due to the presence of different biomolecules in the Partheniumleaf extract capping the silver nanoparticles efficiently blocking them from sensing any analyte.
[00097] Melamine sensing based on on-chip interference biosynthesis was compared with off-chip interference biosynthesis at 1 ppm of melamine using TECAN based absorbance studies and the results have been plotted as two and three dimensional graphs as shown in FIGs. 10A-B. As shown in FIG. 11A, an optical setup having a laser diode light source with a photo detector (OPT 101) was utilized for detecting melamine by interference based sensing. As shown in FIG. 11B, a graph was plotted using the above said setup and the variation was observed between control and melamine of 0.1, 1, 10, 100 and 1000 ppm concentration at different time variation. Selectivity test was carried out using different possible interferents at 100 ppm concentration along with melamine and control. There was no noticeable color change observed for other analytes being used in the selectivity test except for melamine and the results have been produced in FIGs. 12A-B.
[00098] It was identified that parthenin and caffeic acid (the major phytochemical components in Partheniumleaf extract) were interacted with melamine through strong hydrogen-bonding interaction. Consequently, the formation of silver nanoparticles was interrupted by melamine since there was no enough reducing agent for the reduction of silver ions. As shown in FIG.4B, as the concentration of melamine increases there was absorbance decrease in the LSPR of silver nanoparticles at 414 nm and an increase in absorbance at 600 nm. FIG. 13 illustrates the mechanism of melamine interruption in the biosynthesis of silver nanoparticles using Partheniumhisterophorus leaf extract as reducing agent.

ADVANTAGES OF THE PRESENT INVENTION
[00099] The present disclosure provides a portable device that enables real time detection of target analytes in test samples.
[000100] The present disclosure provides a portable device that has precise control over shape and size distribution of resulting metal nanoparticles, and thereby enabling effective and accurate detection of target analytes.
[000101] The present disclosure provides a portable device that facilitates interference based detection of target analyte using plant extract as reducing agent and is completely devoid of hazardous chemicals, and thereby making the detection method safe and environment friendly.
[000102] The present disclosure provides a device that requires very less amount of metal precursor, reducing agent and test sample for the detection of target analyte in the test sample.
[000103] The present disclosure provides a portable device that is easy to operate, energy efficient and allowing different reactions to take place at the same time.
[000104] The present disclosure provides a portable device that can facilitate accurate detection of target analytes with relatively lower test sample volumes than known systems.
[000105] The present disclosure provides a portable device for detection of analytes that overcomes the drawbacks of the prior art.
[000106] The present disclosure provides a colorimetric method for detection of target analytes that is simple, rapid and highly accurate.