WO2020008469A1

WO2020008469A1 - An integrated opto-microfluidic platform for real-time detection of gases in biosamples and liquids

Info

Publication number: WO2020008469A1
Application number: PCT/IN2018/050641
Authority: WO
Inventors: Ashis Kumar SEN
Original assignee: INDIAN INSTITUTE OF TECHNOLOGY MADRAS (IIT Madras)
Priority date: 2018-07-03
Filing date: 2018-10-09
Publication date: 2020-01-09

Abstract

The present invention relates to an integrated opto-microfluidic device for real-time detection of gases in biosamples and liquids. The device comprises two different modules, a particle separation module and an optical detection module, where the particle free sample (blood) is separated from particles (blood cells) present in the original sample (blood sample)and infused into the detection module. The particle-free sample reacts with a sensitive chemical probe to provide a fluorescence (or other optical) signal upon excitation using a light source such as laser, and the signal is detected using suitable optoelectronics.

Description

DESCRIPTION

TITLE OF THE INVENTION

AN INTEGRATED OPTO-MICROFLUIDIC PLATFORM FOR REAL-TIME DETECTION OF GASES IN BIOSAMPLES AND LIQUIDS

FIELD OF THE INVENTION

The present invention relates to an integrated opto-microfluidic device for fluorescence or optical signal based real-time detection or continuous monitoring of gases in biosamples and other liquids.

BACKGROUND OF THE INVENTION

The endogenous signaling molecules such as H₂S,CO and NO (also called as gasotransmitters), play a vital role in the pathophysiological conditions via cellular signaling pathways [A.K. Mustafa et al, Science signaling, 2, 2009; A. Hermann et al., Springer, New York, NY, 2012] In addition, hydrogen peroxide (H2O2), although not considered as a gasotransmitters, plays a major role in signaling and pathophysiology [A.K. Mustafa et al., Science signaling, 2, 2009] There is a huge demand for detection of gasotransmitters in blood and other biosamples (synovial fluid, cerebrospinal fluid, etc.) in the field of healthcare diagnosis and prognosis. The concentration of gasotransmitters in blood can be used for the detection and monitoring of a wide variety of pathological conditions such as Alzheimer’s disease, enthylmalonic encephalopathy, down syndrome, reno-vascular hypertension, vascular endothelial dysfunction, cardiac ischemia disease, microcirculation in the brain, systemic inflammatory response syndrome (SIRS) and sepsis [A.K. Mustafa et al, Science signaling, 2, 2009; A. Hermann et al, Springer, New York, NY, 2012; M. Whiteman et al., Expert Rev. Clin. Pharmacol. 2011, 4, 13-32; Eto K et al., Biochem Biophys Res Commun, 2002, 293, 1485-1488; Geng B et al, Biochem Biophys Res Commun, 2004, 318, 756-763] Similarly, concentration of gasotransmitters in synovial fluid can indicate osteoarthritis and rheumatoid arthritis [P.K. Moore et al, Springer, 2015] Periodic or continuous monitoring of gasotransmitter levels in human blood (and other bio-samples) can prove indispensable for the diagnosis, prognosis and management of pathophysiological conditions. In addition, detection of one or more of these gases (H₂S, CO, NO and H2O2) has relevance in various other fields such as in the analysis of crude oil, water and food and beverages, for quality monitoring [Heshka, N.E et al., J. Vis. Exp. (106), e534l6; M. O. Gorbunovaa et al, Journal of Analytical Chemistry, 2017, 72(12), 1263-1269] For example, measurement of ¾S level in crude oil is important due to its high toxicity and for safety requirements in the event of a release or spill [Heshka, N.E et al, J. Vis. Exp. (106), e534l6]. Similarly, presence of ¾S in water can indicate acute oxygen deficiency [M. O. Gorbunovaa et al, Journal of Analytical Chemistry, 2017, 72(12), 1263-1269] and fecal contamination [ Gupta S et al., J Appl Microbiol, 2008, 104, 388-395] The presence of H₂S and H2O2 in beverages such as wine has been linked to wine spoilage and thus can impact large economic losses [Hao Wang et al, Journal of Food Science, 2018, 83(1); Julien Heritier et al, Food Chemistry, 2016, 211, 957-962] The measurement of ¾S and H₂q2ίh milk is essential for meeting safety requirements [Masoud Shariati-Rad et al., RSC Adv., 2017, 7, 28626-28636; Martin NH et al., J Food Prot, 2014, 77(10), 1809-1813]

There are a number of techniques available to quantify the amount of the above gases in liquids, such as gas chromatography (GC) [Masoud Shariati-Rad et al, RSC Adv., 2017, 7, 28626], colorimetric [D. Jimenez et al., J. Am. Chem. Soc., 2003, 125, 9000-9001], electrochemical assays [D. G. Searcy et al., Anal. Biochem., 2004, 324, 269-275], ion selective electrode method [Doeller JE et al, Anal Biochem, 2005, 341, 40-51] and chemiluminescence [W. J. Cooper et al., Mar. Chem., 2000, 70, 191-200] However, such methods require tedious and complicated sample preparation and most of the methods are operationally complex, costly, and time consuming. These issues can be potentially eliminated by employing microfluidics technology that offers unique advantages in terms of low sample volume, faster response and integration of sample preparation and detection modules [E.K. Sackmann et al, Nature. 2014, 507, 181-189]

Microfluidic devices for the detection of gases in biosamples and liquids can be broadly classified, based on the detection or measurement technique, into two categories: electrochemistry and opto-fluidics (either by absorbance, colorimetry or fluorimetry). Several works have been reported on the measurement of nitric oxide in buffer solutions [ W. Cha et al, Anal. Chem. 2010, 82, 3300-3305], released from (single) cells [S.T. Halpin et al., Anal. Chem. 2010, 82, 7492-7497; E.C. Metto et al., Anal. Chem. 2013, 85, 10188-10195] and m other bio- fluids [S. Jiang et al, Nat. Commun. 2013, 4, 1-7; R.A. Hunter, Anal. Chem. 2013, 85, 6066- 6072 and US 9,201,037 B2] using microfluidic platforms. Most of the works are based on electrochemistry in which for a given potential difference, current is generated between a functional and reference electrodes, when there is presence of nitric oxide [W. Cha et al., Anal. Chem. 2010, 82, 3300-3305] In this case, the functional electrode is specific to the species of interest. Similar attempts were made to miniaturise the detection of hydrogen sulphide or bisulfide in effluents of microdialysis [X. Zhu et al., Biosens. Bioelectron. 2014, 55, 438-445] and cerebrospinal fluid based on absorbance [F. Gu et al., Analyst. 2015, 140, 3814-3819] However, here, the detection was carried out in a PTFE tube outside the microfluidic device due to the limitations in the channel design.

A comprehensive review of the literature clearly indicates that an integrated microfluidic device capable of sample preparation (for example blood-plasma separation and mixing of chemical probe with sample/blood-plasma) and on-chip optical detection of FFS, FFCF, CO and NO has not been developed yet. Among the various detection methods, flourimetry provides higher sensitivity, simplicity, real-time and non-destructive detection of biological samples with good temporal and spatial resolution [N. Dufton et al, Sci. Rep. 2012, 2,1-11]

Thus the present invention provides an integrated opto-microfluidic platform for fluorescence or other optical signal based real-time detection or continuous monitoring of gases including FFS, FFCF, CO and NO in biosamples and other liquids. The platform integrates a separation module with fluorescence or other optical signal based detection module. The particle-free sample reacts with a sensitive chemical probe to provide fluorescence or other optical signal upon excitation using a light source such as laser, and the signal is detected using suitable optoelectronics.

SUMMARY OF THE INVENTION

The present invention is relates to an integrated opto-microfluidic device for fluorescence or other optical signal based real-time detection or continuous monitoring of gases including H₂S, H₂0₂, CO and NO in biosamples and other liquids.

In one embodiment, the present invention provides a device that integrates a separation module with fluorescence or other optical signal based detection module, where the particle-free sample reacts with a sensitive chemical probe to provide fluorescence or other optical signal upon excitation by a light source such as laser, and the signal is detected using suitable optoelectronics.

In another embodiment, the proposed device comprises two different modules, a particle separation module and an optical detection module. In the particle separation module, particle- free sample (such as plasma) is separated from particles (such as blood cells) present in the original sample (blood sample) and infused into the detection module.

The detection module comprises mixing and detection zones, wherein the particle-free sample (such as cell-free plasma) is mixed in the mixing zone with a suitable chemical probe in a serpentine channel (or a channel of any other configuration) that provides the channel length (or residence time for the two liquids) required for complete mixing. In the detection zone, the mixture of the gas and chemical probe is excited with light (laser) at a particular wavelength. Optical fibers (or waveguides or any other suitable arrangement) placed on the device guide the light from the laser source to the detection zone. The emitted fluorescence signal is collected using optical fibres (or waveguides or any other suitable arrangement) and recorded using a high- speed and highly sensitive photodetector. The intensity of the fluorescence signal is representative of the concentration of the gas present in the sample.

In another preferred embodiment, the present invention shows that the proposed device can be used for the detection biomarkers (such as procalcitonin) in biosamples. Depending on the application, the proposed platform can be suitably modified. The platform can also be used with particle-free liquid (such as plasma) obtained using centrifugation (or by any other means) and in that case the separation module is not required and the particle-free sample can be directly infused into the detection module. Similarly, a liquid sample can be directly infused directly into the detection module (without requiring the separation module) although in that case presence of particles would significantly affect the optical measurements.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Schematic diagram of the proposed integrated opto-microfluidic device for real-time detection of gases in biosamples and other liquids. Figure 2 (a) Variation of fluorescent photon counts (intensity, I) with spiked concentration of H₂S in blood-plasma, (b) Normalized fluorescent intensity variation with spiked concentration of H₂S in blood-plasma. The solid line shows the linear fit of the data with R² = 0.99.

Referring to the drawings, the embodiments of the present invention are further described. The figures are not necessarily drawn to scale, and in some instances the drawings have been exaggerated or simplified for illustrative purposes only. One of ordinary skill in the art may appreciate the many possible applications and variations of the present invention based on the following examples of possible embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

The following description is presented to enable any person skilled in the art to make and use the embodiments, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present disclosure. Thus, the present invention is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

The present invention relates to a device that comprises two different modules, a particle separation module and an optical detection module. In the particle separation module, particle- free sample (plasma) is separated from particles (blood cells) present in the original sample (blood sample) and infused into the detection module.

The detection module comprises mixing and detection zones. Microfluidic channels of dimensions ranging from tens of microns to hundreds of microns form the flow path in both the modules. In the mixing zone, particle-free sample (cell-free plasma) is mixed with a suitable chemical probe in a serpentine channel (or a channel of any other configuration) that provides the channel length (or residence time for the two liquids) required for complete mixing. Alternatively, instead of the serpentine channel (or channel of any other configuration), the mixture of the gasotransmitter and chemical probe can been capsulated in the form of micro droplets in a suitable immiscible phase (oil) to enhance mixing due to smaller length scale of micro droplets. The chemical probe is selective to the specific gas which is to be detected or monitored. The chemical probe is selected from any one of the following;- rhodamine, dansyl azide and 7- azido-4-methylcoumarin for H₂S, phenanthroimidazole, benzonitrile and phenylboronate for H2O2, diaminofluoresceine and pyrene based cyclio-quinodimethane for NO, and palladium chloride for CO. The selection of probe is based on high specificity (less or no interference with other compounds), high sensitivity (detection limit <5 mM) and lower response time (<1.0 ms). The chemical reaction between the specific gas to be detected in the sample and the chemical probe yields in a chemical compound that fluoresces upon excitation with light using laser or other light source of a particular wavelength to provide emission at specific wavelength.

In the detection zone, the mixture of the gas and chemical probe is excited with light

(laser) at a particular wavelength. Optical fibers (or waveguides or any other suitable arrangement) placed on the device which guides the light from the laser source to the detection zone. The emitted fluorescence signal is collected using optical fibres (or waveguides or any other suitable arrangement) and recorded using a high-speed and highly sensitive photodetector. The intensity of the fluorescence signal is representative of the concentration of the gas present in the sample.

Fig. 2a and 2b, respectively, show the variation in the fluorescent photon counts and normalized fluorescence intensity detected using the proposed optofluidic platform with concentration of H2S spiked in blood-plasma. The solid line shows the linear fit of the data with R² = 0.99. The fluorescent intensity (I) linearly varies with the addition of sodium sulfide (donor of H2S) and the fluorescent intensity

is normalised with the probe intensity (Io).

Apart from detection of gases in liquids, the proposed device can be used for the detection of biomarkers (such as procalcitonin) in biosamples. Depending on the application, the proposed platform can be suitably modified. The platform can also be used with particle-free liquid (plasma) obtained using centrifugation (or by any other means) and in that case the separation module is not required and the particle-free sample can be directly infused into the detection module. Similarly, a liquid sample can be infused directly into the detection module (without requiring the separation module) although in that case presence of particles would significantly affect the optical measurements. However, applications that require continuous monitoring would necessitate both the modules of the proposed platform where sample (blood sample) can be infused into the system continuously to monitor the concentration of gases in the sample in real-time. The platform can be fabricated using either any one or a combination of different materials such as silicon, glass polymers or metals or any other material based on the compatibility of the materials with the sample and chemical probe used. The chip can be fabricated using micro-milling and lithography or any other fabrication techniques. Optical filters can be used at the detector for selectively detect wavelengths within a specific range of wavelengths depending on the probe and gasotransmitter.

An integrated opto-microfluidic platform for real-time monitoring of gasotransmitters and other gases in biosamples and other liquids has applications in the (1) Detection or continuous monitoring of gasotransmitters in blood and other biosamples. (2) Detection of one or more of the gases () in crude oil, water and food and beverages.

It may be appreciated by those skilled in the art that the drawings, examples and detailed description herein are to be regarded in an illustrative rather than a restrictive manner.

Claims

We Claim:

1. An integrated opto-microfluidic device for real-time detection of gases/gasotransmitters in biosample comprising:

a particle separation module for separating particle-free sample from whole particle containing sample;

an optical detection module integrated with particle separation module, comprises mixing and detection zones, where the particle free sample reacts with a sensitive chemical probe providing fluorescence signal upon excitation;

wherein the intensity of the fluorescence signal represents the concentration of the gas present in the sample.

2. The integrated opto-microfluidic device as claimed in claim 1, wherein the detection of gasotransmitters includes H₂S, H₂0₂, CO and NO.

3. The integrated opto-microfluidic device as claimed in claim 1, wherein the biosample is blood sample.

4. The integrated opto-microfluidic device as claimed in claim 1, wherein the particle-free sample is plasma from blood cells

5. The integrated opto-microfluidic device as claimed in claim 1, wherein the mixing zone includes serpentine channel but not limited to different channel configurations that provides the longer channel length for complete mixing.

6. The integrated opto-microfluidic device as claimed in claim 1, wherein the mixing is enhanced by encapsulating the mixture of the gasotransmitter and chemical probe in a suitable immiscible phase.

7. The integrated opto-microfluidic device as claimed in claim 1, wherein the chemical probe is selective to the specific gas.

8. The integrated opto-microfluidic device as claimed in claim 1, wherein the chemical probe for H₂S is rhodamine, dansyl azide and 7-azido-4-methylcoumarin.

9. The integrated opto-microfluidic device as claimed in claim 1, wherein the chemical probe for H₂0₂ is phenanthroimidazole, benzonitrile and phenylboronate.

10. The integrated opto-microfluidic device as claimed in claim 1, wherein the chemical probe for NO is diaminofluoresceine and pyrene based cyclio-quinodimethane.

11. The integrated opto-microfluidic device as claimed in claim 1, wherein the chemical probe for CO is palladium chloride.

12. The integrated opto-microfluidic device as claimed in claim 1, wherein the selection of chemical probe is based on high specificity, high sensitivity and lower response time.

13. The integrated opto-microfluidic device as claimed in claim 1, wherein the reaction of the gas in the sample and chemical probe emits fluoresces upon excitation with light.

14. The integrated opto-microfluidic device as claimed in claim 1, wherein the optical fiber is placed in the detection zone for guiding the light source to the detector.

15. The integrated opto-microfluidic device as claimed in claim 1, wherein the emitted fluorescence signal is collected and recorded using a high-speed and highly sensitive photodetector.

16. The integrated opto-microfluidic device as claimed in claim 1, wherein the device detects biomarkers including procalcitonin in biosamples.

17. The integrated opto-microfluidic device as claimed in claim 1, wherein the device uses optical filter for selectively detect wavelengths within a specific range of wavelengths depending on the probe and gasotransmitter.

18. The integrated opto-microfluidic device as claimed in claim 1, wherein the device detects one or more gases present in the crude oil, water and food and beverages.