A Localized surface plasmon resonance (LSPR) sensor integrated automated microfluidic system for multiplex inflammatory biomarker detection.

Inflammation is a complex biological response of the human body to external or internal stimuli, such as invading pathogens, defective cells, or irritating substances. One important indicator of inflammatory conditions or the progress of various diseases, such as cancer, cardiovascular diseases, neurological diseases, connective tissue diseases, sepsis, or Alzheimer's disease, is the concentration level of inflammatory biomarkers, including immunoglobulins, cytokines, and C-reactive protein (CRP). Since inflammatory biomarkers are highly correlated with each other, it is important to measure them simultaneously. To enable continuous and dynamic inflammatory biomarker detection, we utilized localized surface plasmon resonance (LSPR) to perform label-free molecule sensing. Since the LSPR sensing mechanism requires only a small sensing area with simplified optical setup, it can be easily integrated with a microfluidic device. To simplify reagent operation complexity, we developed an automated microfluidic control system to control reagent guiding and switching in the immunoassay with less manual processes and potential operation errors. Our results successfully demonstrated multiplex IgG, TNF-α, and CRP measurement with only 60 µL assay volume and 3.5 h assay time. In each test, 20 sensing spot measurements under four different reagent conditions can be performed. Overall, we envision that the LSPR sensor integrated automated microfluidic control system could perform rapid, multiplex, and multiparallel continuous inflammatory biomarker detection, which would be beneficial for various applications, such as immune status monitoring, diagnosis and prognosis of inflammatory diseases.

Antibiotic Susceptibility Test with Surface-Enhanced Raman Scattering in a Microfluidic System.

Antibiotic susceptibility test (AST) is essential in clinical diagnosis of serious bacterial infection, such as sepsis, while it typically takes 2-5 days for sample culture, antibiotic treatment, and reading result. Detecting metabolites secreted from bacteria with surface-enhanced Raman scattering (SERS) enables rapid determination of antibiotic susceptibility, reducing the AST time to 1-2 days. However, it still requires 1 day of culture time to obtain sufficient quantity of bacteria for sample washing, bacterial extraction, and antibiotic treatment. Additionally, the whole procedure, manually performed in open environment, often suffers from contamination and human error. To address the above problems, a microfluidic system integrating membrane filtration and the SERS-active substrate (MF-SERS) was developed to perform on-chip bacterial enrichment, metabolite collection, and in situ SERS measurements for antibiotic susceptibility test. Using Escherichia coli as the prototype bacterium, the lowest SERS detection limit of bacterial concentration of the MF-SERS system is 103 CFU/mL, which is 4 orders of magnitude lower than that using centrifugation-purification procedure, significantly shortening the bacterial culture time. The bacteria and secreted metabolites are enclosed during bacterial trapping, metabolite filtration, and SERS detection, thus minimizing possible contamination and human errors. Finally, the successful demonstration of AST on E. coli with a concentration of 103 CFU/mL is presented. Overall, the MF-SERS system with a miniature size and well-confined microenvironment allows the integration of multiple bacteria processes for bacterial enrichment, culture, and determination of AST.

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Salivary biomarker combination prediction model for the diagnosis of periodontitis in a Taiwanese population.

BACKGROUND/PURPOSE: This study aimed at screening the diagnostic potential of salivary biomarkers and pairing them to establish a prediction model with higher accuracy in diagnosing periodontitis in the Taiwanese population. METHODS: Fifty-seven participants were divided into a non-periodontitis group and a periodontitis group. Salivary biomarkers CRP, IL-6, IL-8, IL-1ß, IL-1ra, lactoferrin, MMP-8, MMP-9, PDGF-BB, TNF-α, and VEGF, were analyzed. The potential and reliability of the biomarkers for diagnosing periodontitis were analyzed dichotomously. The correlation of individual biomarkers with periodontitis was assessed using the Spearman rank correlation coefficient with logistic regression. The combinational prediction model was evaluated using the area under the receiver operating characteristic curve (AUC). RESULTS: Regarding individual biomarkers, IL-1ß and MMP-9 levels were significantly higher, and TNF-α was significantly lower in the periodontitis group. IL-1ß, MMP-8, and MMP-9 exhibited a greater odds ratio with statistical significance in the dichotomous table. The combination of three biomarkers yielded AUCs of 0.821-0.853, and the combination of IL-1ß, IL-1ra, and MMP-9 exhibited the highest AUC (0.853), with high sensitivity (73.3%) and specificity (88.9%). CONCLUSION: Regarding individual biomarkers, IL-1ß, MMP-8, and MMP-9 showed potential for identifying patients with periodontitis. The combination of IL-1ß, IL-1ra, and MMP-9 might be feasible for developing a future point-of-care device for diagnosing periodontitis.

Pre-Clinical Tests of an Integrated CMOS Biomolecular Sensor for Cardiac Diseases Diagnosis.

Coronary artery disease and its related complications pose great threats to human health. In this work, we aim to clinically evaluate a CMOS field-effect biomolecular sensor for cardiac biomarkers, cardiac-specific troponin-I (cTnI), N-terminal prohormone brain natriuretic peptide (NT-proBNP), and interleukin-6 (IL-6). The CMOS biosensor is implemented via a standard commercialized 0.35 µm CMOS process. To validate the sensing characteristics, in buffer conditions, the developed CMOS biosensor has identified the detection limits of IL-6, cTnI, and NT-proBNP as being 45 pM, 32 pM, and 32 pM, respectively. In clinical serum conditions, furthermore, the developed CMOS biosensor performs a good correlation with an enzyme-linked immuno-sorbent assay (ELISA) obtained from a hospital central laboratory. Based on this work, the CMOS field-effect biosensor poses good potential for accomplishing the needs of a point-of-care testing (POCT) system for heart disease diagnosis.

Dual Ion-Selective Membrane Deposited Ion-Sensitive Field-Effect Transistor Integrating a Whole Blood Processing Microchamber for In Situ Blood Ion Testing.

Blood ion testing is one of the methods that is commonly used for monitoring the immune status and providing physiological information for disease diagnosis. However, traditional blood ion sensing methods often require well-trained operators to process the whole blood sample and perform the measurements using bulky instruments, making real-time and continuous blood ion sensing at the bedside difficult. To address the above issues, we proposed a dual ion-selective membrane deposited ion-sensitive field-effect transistor (DISM-ISFET) sensor integrating a microchamber to enable on-chip serum extraction and in situ Na+/K+ ion sensing. As a proof of concept, we sequentially dispensed NaCl and KCl solutions at various concentrations on the DISM-ISFET to find out the highest ion sensitivity and selectivity. Next, we also confirm the high red blood cell sedimentation and serum purity using a microchamber. Finally, we evaluated the system performance using nine clinical whole blood samples and compared their Na+/K+ ion-sensing results with a commercial pocket ion meter. To sum up, our results showed that a DISM-ISFET system can successfully extract 200 µL serum from 500 µL whole blood sample and simultaneously achieve Na+/K+ ion sensing. All the sample processes and measurements can be finished within 10 min in a single chip. We envision this compact and easy-to-use system can be potentially used for various medical environments requiring real-time and continuous blood ion monitoring, such as in a hemodialysis room, operation room, and intensive care unit.

Deep Learning-Assisted Surface-Enhanced Raman Scattering for Rapid Bacterial Identification.

Bloodstream infection (BSI) is characterized by the presence of viable microorganisms in the bloodstream and may induce systemic immune responses. Early and appropriate antibiotic usage is crucial to effectively treating BSI. However, conventional culture-based microbiological diagnostics are time-consuming and cannot provide timely bacterial identification for subsequent antimicrobial susceptibility test (AST) and clinical decision-making. To address this issue, modern microbiological diagnostics have been developed, such as surface-enhanced Raman scattering (SERS), which is a sensitive, label-free, and quick bacterial detection method measuring specific bacterial metabolites. In this study, we aim to integrate a new deep learning (DL) method, Vision Transformer (ViT), with bacterial SERS spectral analysis to build the SERS-DL model for rapid identification of Gram type, species, and resistant strains. To demonstrate the feasibility of our approach, we used 11,774 SERS spectra obtained directly from eight common bacterial species in clinical blood samples without artificial introduction as the training dataset for the SERS-DL model. Our results showed that ViT achieved excellent identification accuracy of 99.30% for Gram type and 97.56% for species. Moreover, we employed transfer learning by using the Gram-positive species identifier as a pre-trained model to perform the antibiotic-resistant strain task. The identification accuracy of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA) can reach 98.5% with only 200-dataset requirement. In summary, our SERS-DL model has great potential to provide a quick clinical reference to determine the bacterial Gram type, species, and even resistant strains, which can guide early antibiotic usage in BSI.

A Microfluidic Platform with an Embedded Miniaturized Electrochemical Sensor for On-Chip Plasma Extraction Followed by In Situ High-Sensitivity C-Reactive Protein (hs-CRP) Detection.

Blood testing is a clinical diagnostic tool to evaluate physiological conditions, the immune system response, or the presence of infection from whole blood samples. Although conventional blood testing can provide rich biological information, it usually requires complicated and tedious whole blood processing steps operated by benchtop instruments and well-experienced technicians, limiting its usage in point-of-care (POC) settings. To address the above problems, we propose a microfluidic platform for on-chip plasma extraction directly from whole blood and in situ biomarker detection. Herein, we chose C-reactive protein (CRP) as the target biomarker, which can be used to predict fatal cardiovascular disease (CVD) events such as heart attacks and strokes. To achieve a rapid, undiluted, and high-purity on-chip plasma extraction, we combined two whole blood processing methods: (1) anti-D immunoglobulin-assisted sedimentation, and (2) membrane filtration. To perform in situ CRP detection, we fabricated a three-dimensional (3D) microchannel with an embedded electrochemical (EC) sensor, which has a modular design to attach the blood collector and buffer reservoir with standard Luer connectors. As a proof of concept, we first confirmed that the dual plasma extraction design achieved the same purity level as the standard centrifugation method with smaller sample (100 µL of plasma extracted from 400 µL of whole blood) and time (7 min) requirements. Next, we validated the functionalization protocol of the EC sensor, followed by evaluating the detection of CRP spiked in plasma and whole blood. Our microfluidic platform performed on-chip plasma extraction directly from whole blood and in situ CRP detection at a 0.1-10 µg/mL concentration range, covering the CVD risk evaluation level of the high-sensitivity CRP (hs-CRP) test. Based on the above features, we believe that this platform constitutes a flexible way to integrate the processing of complex samples with accurate biomarker detection in a sample-to-answer POC platform, which can be applied in CVD risk monitoring under critical clinical situations.

An antibiotic concentration gradient microfluidic device integrating surface-enhanced Raman spectroscopy for multiplex antimicrobial susceptibility testing.

Antimicrobial susceptibility testing (AST) is a key measure in clinical microbiology laboratories to enable appropriate antimicrobial administration. During an AST, the determination of the minimum inhibitory concentration (MIC) is an important step in which the bacterial responses to an antibiotic at a series of concentrations obtained in separate bacterial growth chambers or sites are compared. However, the preparation of different antibiotic concentrations is time-consuming and labor-intensive. In this paper, we present a microfluidic device that generates a concentration gradient for antibiotics that is produced by diffusion in the laminar flow regime along a series of lateral microwells to encapsulate bacteria for antibiotic treatment. All the AST preparation steps (including bacterium loading, antibiotic concentration generation, buffer washing, and isolated bacterial growth with an antibiotic) can be performed in a single chip. The viable bacterial cells in each microwell after the antibiotic treatment are then quantified by their surface-enhanced Raman scattering (SERS) signals that are acquired after placing a uniform SERS-active substrate in contact with all the microwells. For proof-of-concept, we demonstrated the AST performance of this system on ampicillin (AMP)-susceptible and -resistant E. coli strains. Compared with the parameters for conventional AST methods, the AST procedure based on this chip requires only 20 µL of bacteria solution and 5 h of operation time. This result indicates that this integrated system can greatly shorten and simplify the tedious and labor-intensive procedures required for current standard AST methods.

Biosensing Amplification by Hybridization Chain Reaction on Phase-Sensitive Surface Plasmon Resonance.

Surface Plasmon Resonance (SPR) is widely used in biological and chemical sensing with fascinating properties. However, the application of SPR to detect trace targets is hampered by non-specific binding and poor signal. A variety of approaches for amplification have been explored to overcome this deficiency including DNA aptamers as versatile target detection tools. Hybridization chain reaction (HCR) is a high-efficiency enzyme-free DNA amplification method operated at room temperature, in which two stable species of DNA hairpins coexist in solution until the introduction of the initiator strand triggers a cascade of hybridization events. At an optimal salt condition, as the concentrations of H1 and H2 increased, the HCR signals were enhanced, leading to signal amplification reaching up to 6.5-fold of the detection measure at 30 min. This feature enables DNA to act as an amplifying transducer for biosensing applications to provide an enzyme-free alternative that can easily detect complex DNA sequences. Improvement of more diverse recognition events can be achieved by integrating HCR with a phase-sensitive SPR (pSPR)-tested aptamer stimulus. This work seeks to establish pSPR aptamer system for highly informative sensing by means of an amplification HCR. Thus, combining pSPR and HCR technologies provide an expandable platform for sensitive biosensing.

A microfluidic microwell device operated by the automated microfluidic control system for surface-enhanced Raman scattering-based antimicrobial susceptibility testing.

Bloodstream infection (BSI) is a serious public health issue worldwide. Timely and effective antibiotics for controlling infection are crucial towards patient outcomes. However, the current culture-based methods of identifying bacteria and antimicrobial susceptibility testing (AST) remain labor-intensive and time-consuming, and are unable to provide early support to physicians in critical hours. To improve the effectiveness of early antibiotic therapy, Surface-enhanced Raman scattering (SERS) technology, has been used in bacterial detection and AST based on its high specificity and label-free features. To simplify sample preparation steps in SERS-AST, we proposed an automated microfluidic control system to integrate all required procedures into a single device. Our preliminary results demonstrated the system can achieve on-chip reagent replacement, bacteria trapping, and buffer exchange. Finally, in-situ SERS-AST was performed within 3.5 h by loading isolates of ampicilin susceptible and resistant E. coli and clear discrimination of two strains under antibiotic treatment was demonstrated. Overall, our system can standardize and simplify the SERS-AST protocol and implicate parallel bacterial detection. This prototypical integration demonstrates timely microbiological support to optimize early antibiotic therapy for fighting bacteremia.

Enhancing Raman signals from bacteria using dielectrophoretic force between conductive lensed fiber and black silicon.

An osmium-coated lensed fiber (OLF) probe combined with a silver-coated black silicon (SBS) substrate was used to generate a dielectrophoretic (DEP) force that traps bacteria and enables Raman signal detection from bacteria. The lensed fiber coated with a 2-nm osmium layer was used as an electrode for the DEP force and also as a lens to excite Raman signals. The black silicon coated with a 150-nm silver layer was used both as the surface-enhanced Raman scattering (SERS) substrate and the counter electrode. The enhanced Raman signal was collected by the same OLF probe and further analyzed with a spectrometer. For Raman measurements, a drop of bacterial suspension was placed between the OLF probe and the SBS substrate. By controlling the frequency of an AC voltage on the OLF probe and SBS substrate, a DEP force at 1 MHz concentrated bacteria on the SBS surface and removed the unbound micro-objects in the solution at 1 kHz. A bacteria concentration of 6 × 104 CFU/mL (colony forming units per mL) could be identified in less than 15 min, using a volume of only 1 µL, by recording the variation of the Raman peak at 740 cm-1.

Multiplexed spectral signature detection for microfluidic color-coded bioparticle flow.

Here, we report a high-speed photospectral detection technique capable of discriminating subtle variations of spectral signature among fluorescently labeled cells and microspheres flowing in a microfluidic channel. The key component used in our study is a strain-tunable nanoimprinted grating microdevice coupled with a photomultiplier tube (PMT). The microdevice permits acquisition of the continuous spectral profiles of multiple fluorescent emission sources at 1 kHz. Optically connected to a microfluidic flow chamber via a multimode optical fiber, our multiwavelength detection platform allows for cytometric measurement of cell groups emitting nearly identical fluorescence signals with a maximum emission wavelength difference as small as 5 nm. The same platform also allows us to demonstrate microfluidic flow cytometry of four different microsphere types in a wavelength bandwidth as narrow as 40 nm at a high (>85%) confidence level. Our study shows that detection of fluorescent spectral signatures at high speed and high resolution can expand specificity of multicolor flow cytometry. The enhanced capability enables multiplexed analysis of color-coded bioparticles based on single-laser excitation and single-detector spectroscopy in a microfluidic setting. The fluorescence signal discrimination power achieved by the optofluidic technology holds great promise to enable quantification of cellular parameters with higher accuracy as well as enumeration of a larger number of cell types than conventional flow cytometric methods.

Recent advancements in microfluidics that integrate electrical sensors for whole blood analysis.

Whole blood analysis reveals crucial information about various physiological and pathological conditions, including cancer metastasis, infection, and immune status, among others. Despite this rich information, the complex composition of whole blood usually required multiple sample preparation steps to purify targeted analytes. Traditionally, whole blood preparation processes, including centrifugation, lysis, dilution, or staining, are usually manually operated by well-trained technicians using bench-top instruments. This preparation can require a large blood volume and cannot be directly integrated with detection systems. Recently, various studies have integrated microfluidics with electrical sensors for whole blood analysis, with a focus on cell-based analysis, such as cell type, number, morphology, phenotype, and secreted molecules. These miniaturized systems require less sample and shorter reaction times. Besides, the sample processing and analysis can be fully integrated and automated with minimal operations. We believe these systems can transfer the current whole blood analysis from hospitals or laboratories into clinics or home settings to enable real-time and continuous health condition monitoring in point-of-care settings.

Review of Integrated Optical Biosensors for Point-Of-Care Applications.

This article reviews optical biosensors and their integration with microfluidic channels. The integrated biosensors have the advantages of higher accuracy and sensitivity because they can simultaneously monitor two or more parameters. They can further incorporate many functionalities such as electrical control and signal readout monolithically in a single semiconductor chip, making them ideal candidates for point-of-care testing. In this article, we discuss the applications by specifically looking into point-of-care testing (POCT) using integrated optical sensors. The requirement and future perspective of integrated optical biosensors for POC is addressed.

Bacteria encapsulation and rapid antibiotic susceptibility test using a microfluidic microwell device integrating surface-enhanced Raman scattering.

The antibiotic susceptibility test (AST) is a general laboratory procedure for bacterial identification and characterization and can be utilized to determine effective antimicrobials for individual patients. Due to the low bacterial concentration, conventional AST usually requires a prolonged bacterial culture time and a labor-intensive sample pretreatment process. Therefore, it cannot perform timely diagnosis or treatment, which results in a high mortality rate for seriously infected patients. To address this problem, we developed a microfluidic microwell device integrating surface-enhanced Raman scattering (SERS) technology, or the so called the Microwell-SERS system, to enable a rapid and high-throughput AST. Our results show that the Microwell-SERS system can successfully encapsulate bacteria in a miniaturized microwell with a greatly increased effective bacteria concentration, resulting in a shorter bacterial culture time. By attaching a microchannel onto the microwell, a smooth liquid and air exchange can purify the surrounding buffer and isolate bacteria in an individual microwell for independent SERS measurement. For proof-of-concept, we demonstrated a 2 h AST on susceptible and resistant E. coli and S. aureus with a concentration of 103 CFU mL-1 in the Microwell-SERS system, whereas the previous SERS-AST method required 108 CFU mL-1 bacterial suspension droplets dispensing on a SERS substrate. Based on the above features, we envision that the Microwell-SERS system could achieve highly sensitive, label-free, bacteria detection and rapid AST to enable timely and accurate bacterial infection disease diagnosis.

A microfluidic device for in situ fixation and super-resolved mechanosensation studies of primary cilia.

The primary cilium plays an important role in mechanosensation in mammalian cells. To understand mechanosensation in the primary cilium, we combined a microfluidic device with super-resolution microscopy to study the primary cilium phenotypes. The microfluidic system enabled the precise control of the flow shear within a well-confined cell-culture environment. In addition, in situ cilia fixation was possible by switching from the culture medium to the fixation buffer instantaneously, which preserved the real-time cilium phenotype under the flow shear. After fixation, multiple cilium-specific proteins were immunostained to quantify the cilia bending behavior. We found that >50% of the primary cilia of mouse inner medullary collecting duct cells were highly aligned with the direction of flow under 11 Pa shear stress. Finally, we used super-resolution microscopy to observe the redistribution of two major cilium-specific proteins under flow shear, acetylated alpha-tubulin, and intraflagellar transport protein 88. To the best of our knowledge, this is the first platform to combine a microfluidic device with super-resolution microscopy to enable flow stimulation and in situ fixation for the observation of ciliary protein. This system can potentially be applied to the future development of a stimulation-enabled organ-on-a-chip to observe the intercellular signaling of primary cilia or for the analysis of disease mechanisms associated with ciliary mutations at the organ level.

A Microfluidic Device for Simultaneous Extraction of Plasma, Red Blood Cells, and On-Chip White Blood Cell Trapping.

This study reports a microfluidic device for whole blood processing. The device uses the bifurcation law, cross-flow method, and hydrodynamic flow for simultaneous extraction of plasma, red blood cells, and on-chip white blood cell trapping. The results demonstrate successful plasma and red blood cell collection with a minimum dilution factor (0.76x) and low haemolysis effect. The extracted red blood cells can also be applied for blood type tests. Moreover, the device can trap up to ~1,800 white blood cells in 20 minutes. The three components can be collected simultaneously using only 6 µL of whole blood without any sample preparation processes. Based on these features, the microfluidic device enables low-cost, rapid, and efficient whole blood processing functionality that could potentially be applied for blood analysis in resource-limited environments or point-of-care settings.

A Large-Area Nanoplasmonic Sensor Fabricated by Rapid Thermal Annealing Treatment for Label-Free and Multi-Point Immunoglobulin Sensing.

Immunoglobulins are important biomarkers to evaluate the immune status or development of infectious diseases. To provide timely clinical treatments, it is important to continuously monitor the level of multiple immunoglobulins. Localized surface plasmon resonance (LSPR)-based nanoplasmonic sensors have been demonstrated for multiplex immunoglobulins detection. However, the sensor fabrication process is usually slow and complicated, so it is not accessible for large-area and batch fabrication. Herein, we report a large-area (2 cm × 2 cm) nanofabrication method using physical vapor deposition followed by a rapid thermal annealing treatment. To optimize the sensor performance, we systematically characterized three fabrication conditions, including (1) the deposition thickness; (2) the maximum annealing temperature, and (3) the annealing time. The corresponding absorbance spectrum profile and surface morphology of the nanostructures were observed by a UV-VIS spectrometer and atomic force microscopy. We then tested the sensitivity of the sensor using a glucose solution at different concentrations. The results showed that the sensor with 10 nm gold deposition thickness under 5-min 900 °C rapid thermal annealing can achieve the highest sensitivity (189 nm RIU-1). Finally, we integrated this nanoplasmonic sensor with a microchannel and a motorized stage to perform a 10-spot immunoglobulin detection in 50 min. Based on its real-time, dynamic and multi-point analyte detection capability, the nanoplasmonic sensor has the potential to be applied in high-throughput or multiplex immunoassay analysis, which would be beneficial for disease diagnosis or biomedical research in a simple and cost-effective platform.

A microfluidic device integrating dual CMOS polysilicon nanowire sensors for on-chip whole blood processing and simultaneous detection of multiple analytes.

The hemoglobin-A1c test, measuring the ratio of glycated hemoglobin (HbA1c) to hemoglobin (Hb) levels, has been a standard assay in diabetes diagnosis that removes the day-to-day glucose level variation. Currently, the HbA1c test is restricted to hospitals and central laboratories due to the laborious, time-consuming whole blood processing and bulky instruments. In this paper, we have developed a microfluidic device integrating dual CMOS polysilicon nanowire sensors (MINS) for on-chip whole blood processing and simultaneous detection of multiple analytes. The micromachined polymethylmethacrylate (PMMA) microfluidic device consisted of a serpentine microchannel with multiple dam structures designed for non-lysed cells or debris trapping, uniform plasma/buffer mixing and dilution. The CMOS-fabricated polysilicon nanowire sensors integrated with the microfluidic device were designed for the simultaneous, label-free electrical detection of multiple analytes. Our study first measured the Hb and HbA1c levels in 11 clinical samples via these nanowire sensors. The results were compared with those of standard Hb and HbA1c measurement methods (Hb: the sodium lauryl sulfate hemoglobin detection method; HbA1c: cation-exchange high-performance liquid chromatography) and showed comparable outcomes. Finally, we successfully demonstrated the efficacy of the MINS device's on-chip whole blood processing followed by simultaneous Hb and HbA1c measurement in a clinical sample. Compared to current Hb and HbA1c sensing instruments, the MINS platform is compact and can simultaneously detect two analytes with only 5 µL of whole blood, which corresponds to a 300-fold blood volume reduction. The total assay time, including the in situ sample processing and analyte detection, was just 30 minutes. Based on its on-chip whole blood processing and simultaneous multiple analyte detection functionalities with a lower sample volume requirement and shorter process time, the MINS device can be effectively applied to real-time diabetes diagnostics and monitoring in point-of-care settings.