Microfluidic platform with integrated thermoelectric sensor: applications in biomedical research

Microfluidic platform for immunodetection of biochemical analytes

The review will introduce you to a microfluidic platform with integrated thermoelectric sensor for biomedical application. A significant amount of progress has been achieved in the past decade in the development of more efficient detection technologies for lab-on-a-chip immunoassays that provide increased sensitivity and specificity for point-of-care applications.

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Microfluidics immunosensors offer multiple advantages over the conventional immunoassays that include improved reaction rate, reduced time for incubation of the reactants, and decreased reagents and sample consumption. Moreover, miniaturization and integration of the multiple assay components permit automation, precise fluidic control, increased reproducibility, and the possibility for high-throughput analysis [1].

A wide range of detection technologies has been integrated with lab-on-a-chip biosensors for the quantification of biochemical analytes. The ideal detection method offers high sensitivity coupled with low power consumption, simple fabrication, and minimal sample preparation [2]. Fluorescence, chemiluminescence, and electrochemiluminescence techniques provide detection sensitivities in the ng-pg mL-1 range [3]. Electrochemical immunosensors provide greater sensitivity and versatility in the selection of substrate [4, 5].

Paper-based immunoassays provide rapid and cost-effective techniques for the detection and quantification of molecular species. Some of the applications of paper-based immunoassays include HIV [6] and influenza virus detection [7]. While these detection methods are well-established, each of these techniques has limitations. The major disadvantages of electrochemical detection technology are the effect of external factors such as pH, temperature, and ionic concentration on the performance of the assay [8]. The disadvantages of fluorescent immunosensors are the necessity for highly efficient optical filters and detectors that presents an engineering challenge in the miniaturization of the excitation and detection apparatus without significantly compromising detection sensitivity [9]. Although paper-based microfluidics offers enormous potential for point-of-care devices, the overall sensitivity of the device needs to be improved [10].

Calorimetric microfluidic device principle

To overcome the adversities caused by the detection methods mentioned in the previous section, calorimetric microfluidic platforms have been implemented by various research groups for the detection of and quantification of biochemical processes. An integrated thermoelectric sensor measures the heat released during the enzymatic or chemical reaction and converts it to an electric signal. This technology uses small sample volumes, provides quick response times, and a simplified detection approach. Due to the remarkable common-mode thermal noise rejection capabilities, thin-film thermopiles are suitable for sensing very small temperature changes. Moreover, calorimetry is compatible with most biochemical processes as most of these reactions are exothermic or endothermic and are associated with temperature change.

Microscale thermoelectric devices have been employed to measure temperatures in the range of mK-µK for the characterization of the thermodynamic properties of biomolecules [11] and metabolites measurement [12]. Thermoelectric sensors have been successfully used for the quantification of TNF- α [3], modified nucleobases [13], monitoring the metabolic rate of bacteria [14], detection of enzymatic reactions [15], measurement of DNA and protein binding affinity [16], detection of nucleotide incorporation [17], and DNA hybridization events [18]. Because the calorimetric detection method is universal, it can be applied for assays that cannot be performed using other techniques [19].

In an article published by Saif Mohammad Ishraq Bari et al. (2019), the authors show that a microfluidic device with an integrated thin-film thermopile can be used for sensitive immunodetection of inflammatory cytokines. The sensor is small in size (8mm×8 mm), light in weight, consists of 60 antimony /bismuth thermocouple junctions. The thermopile offers rapid response time and provides a self-generating signal that does not require external power. The sensor operates without any control of external temperature because of its high common-mode thermal noise rejection ratio. The demonstrated microfluidics platform employs a control of the flow rates, maintaining a consistent fluid velocity for both inlets.

Calorimetric immunosensing principle

Calorimetric quantification of TNF- α was successfully demonstrated in a microfluidic device with an integrated thin-film Sb/Bi thermoelectric sensor with a theoretical Seebeck coefficient of 7.14 μV mK-1. The technique is based on sandwiched immunodetection using a pair of matched antibodies that recognize different epitope sites of the TNF- α cytokine. The antibody/analyte complex was immobilized within the measuring junctions of the thermoelectric sensor. Fixed concentration of glucose (55 mM) was injected through a sample loop into the fluid flowing within the microfluidic device. The oxidation of glucose to gluconic acid by glucose oxidase generates 79 kJ mol-1 of energy [21].

Experimental study for quantification of cytokines in cell culture media

The feasibility of the developed novel microfluidic technology was demonstrated via accurate measurement of the concentration of TNF-α in astrocyte cell culture media. The immunoassay was performed in a microfluidic device with an integrated antimony/bismuth thermopile sensor and had a limit of detection of 14 pg mL−1. The device was fabricated using the rapid prototyping xurography technique and consisted of two inlets and a single outlet. Anti-TNF- α monoclonal antibody was used to capture the analyte while the detection was performed using glucose oxidase- conjugated secondary antibody. Glucose (55 mM) was injected through a sample loop into the fluid flowing within the microfluidic device.

• Learn here how to perform unidirectional medium recirculation for microfluidic cell culture.

A nanovolt meter connected to the thermoelectric sensor recorded the voltage change caused by the enzymatic reaction. A standard calibration curve was created using serial dilutions of synthetic TNF-α (0–2000 pg mL−1) by plotting the area under the curve of the signal versus the concentration of the analyte (Fig. 3).

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The efficacy of the device was evaluated by quantifying TNF-α in the cell culture medium of lipopolysaccharide-stimulated and non-stimulated astrocytes. The concentration of TNF-α in lipopolysaccharide treated cells was 252 pg mL-1 and 251 pg mL-1 for absorbance ELISA and microfluidic immunosensor respectively. The levels of TNF-α in non-treated astrocytes measured using conventional ELISA and the microfluidic device exhibited less than 1% variation. The developed biosensor displayed good selectivity as TNF-α determinations agreed well with the standard immunoassay analysis. The microfluidic immunosensor had lower inter-assay variability in comparison to absorbance-based microplate immunoassay. There was no significant difference between the results obtained by the two methods and therefore the calorimetric microfluidic immunosensor can be reasonably applied for the analysis of biological samples.

3D Multiphysics modeling to optimize design parameters for increased sensitivity

3D numerical modeling of a continuous-flow microfluidic thermoelectric biosensor is developed to identify the critical design parameters that provide the maximum signal output (device sensitivity). The simulation results were compared with the experimental measurements from [3]. The collective effect of multiple factors on the heat distribution within the microchannel and the rate of heat transfer to the sensor were simulated using the “laminar flow”, “transport of the diluted species”, and “heat transfer” modules in COMSOL Multiphysics 5.4. Conjugate heat transfer analysis was performed to identify the optimal parameter combination that provides maximum sensitivity.

The factors that heavily determine the performance characteristics of the thermal microfluidic device include channel height, physical properties of the device materials, inlets flow rates ratio, and the presence of a heat sink in contact with the reference junctions of the thermopile. A detailed study of the optimized device parameters to increase device sensitivity can be found in [20].

Future implications of microfluidics calorimetry in biomedical applications

The novel microfluidic immunosensor with an integrated thin-film thermopile for quantification of cytokines in biological samples offers excellent stability, reproducibility, accuracy, and versatility in the choice of the detection enzyme. The developed calorimetric device can detect low levels of heat and utilizes a glucose oxidase conjugated antibody as a novel method for sensitive detection of biochemical analytes. The calorimetric immunosensor had a low limit of detection of 14 pg mL-1. This novel calorimetric method for quantification of TNF- α could be easily extended for sensitive detection of various analytes in biological samples. The results demonstrated high accuracy of the calorimetric immunoassay when compared with gold standard commercial ELISA microplate reader.

Authors’ contribution

Dr. Saif Mohammad Ishraq Bari & Dr. Gergana Nestorova from the Nestorova Lab from Louisana Tech University.

References