Microfluidic Biosensor Research Articles

Surface Modification of Parylene C Film via Buchwald-Hartwig Amination for Organic Solvent-Compatible and Flexible Microfluidic Channel Bonding.

Surface modification offers an efficient and economical route to installing functional groups on a polymer surface. This work demonstrates that primary amine groups can be introduced onto a polymer surface via Buchwald-Hartwig amination, and the functionalized substrates can be chemically bonded to produce functional microfluidic devices. By activating the CCl bond in commercially used poly(chloro-p-xylylene) (parylene C) by Pd catalyst and substituting Cl with the amine source, the amine groups are successfully installed in a facile and recyclable manner. The substrates can be covalently bonded with each other via amine-isocyanate chemistry, providing much higher bonding strength compared to previous methods based on noncovalent adhesive coatings. As a result, transparent and flexible microfluidic channels can be fabricated that are compatible with organic solvents and high pressure. Retention of amine group reactivity in the channel suggests the potential of this methodology for the surface immobilization of functional molecules for microfluidic reactors and biosensors.

Microfluidics-Based Sensing of Biospecies.

Over the past decades, microfluidic devices based on many advanced techniques have aroused widespread attention in the fields of chemical, biological, and analytical applications. Integration of microdevices with a variety of chip designs will facilitate promising functionality. Notably, the combination of microfluidics with functional nanomaterials may provide creative ideas to achieve rapid and sensitive detection of various biospecies. In this review, focused on the microfluids and microdevices in terms of their fabrication, integration, and functions, we summarize the up-to-date developments in microfluidics-based analysis of biospecies, where biomarkers, small molecules, cells, and pathogens as representative biospecies have been explored in-depth. The promising applications of microfluidic biosensors including clinical diagnosis, food safety control, and environmental monitoring are also discussed. This review aims to highlight the importance of microfluidics-based biosensors in achieving high throughput, highly sensitive, and low-cost analysis and to promote microfluidics toward a wider range of applications.

Biosensing on the Centrifugal Microfluidic Lab-on-a-Disc Platform

Lab-on-a-Disc (LoaD) biosensors are increasingly a promising solution for many biosensing applications. In the search for a perfect match between point-of-care (PoC) microfluidic devices and biosensors, the LoaD platform has the potential to be reliable, sensitive, low-cost, and easy-to-use. The present global pandemic draws attention to the importance of rapid sample-to-answer PoC devices for minimising manual intervention and sample manipulation, thus increasing the safety of the health professional while minimising the chances of sample contamination. A biosensor is defined by its ability to measure an analyte by converting a biological binding event to tangible analytical data. With evolving manufacturing processes for both LoaDs and biosensors, it is becoming more feasible to embed biosensors within the platform and/or to pair the microfluidic cartridges with low-cost detection systems. This review considers the basics of the centrifugal microfluidics and describes recent developments in common biosensing methods and novel technologies for fluidic control and automation. Finally, an overview of current devices on the market is provided. This review will guide scientists who want to initiate research in LoaD PoC devices as well as providing valuable reference material to researchers active in the field.

Label-Free Immunoassay of Carcinoembryonic Antigen by Microfluidic Channel Biosensor Based on Imaging Ellipsometry and Its Clinic Application

This study represents a label-free immunoassay for carcinoembryonic antigen (CEA) by microfluidic biosensor based on imaging ellipsometry (BIE) and its application is further validated by clinic sera. The sandwich format of analysis configuration was adopted, in which Rat-mAb was served as capture antibody, rabbit anti-CEA polyclonal antibody (rabbit-pAb) as detection antibody and goat anti-rabbit IgG (goat-IgG) as an enhancer added to increase detection signal. The methodology includes rat-mAb concentration screening, rabbit-pAb and goat-IgG concentration optimization, creation of dose-response curve and determination of cutoff value. Results show the linear range is from 4.0–64.0 ng/ml with a lower limit of 1.0 ng/ml (S/N = 3). Application of the BIE to clinic samples demonstrated that the relation coefficient is 0.94 and substantial agreement with the results of ECLIA’s (Kappa = 0.733, U = 4.35 > U0.01). The proposed method exhibits the potential for application.

Rapid and label-free optical assay of S-layer protein with high sensitivity using TiO2-coated porous silicon-based microfluidic biosensor

S-layer protein (SLP) is a surface protein present in many bacteria and shows unique self-assemble ability for various applications, but its detection methods are very limited. In this paper, a label-free, highly selective and sensitive assay of SLP was developed by using the TiO2-coated porous silicon (PSi)-based microfluidic biosensor. The PSi-based biosensor could be easily prepared in a microfluidic device to build a microfluidic biosensing system for measuring SLP in samples rapidly. As the detection principle of the biosensor was based on the light reflection interference, so there was no labeling requirement. The specific recognition capability of the PSi-based biosensor was achieved by the non-covalently immobilized anti-SLP on the PSi surface. The developed biosensing system showed a rapid response time (5 min) and an excellent specificity to SLP, and displayed a broad detection linear range of 0.001 ∼ 10 μg/mL and a low detection limit of 3.2 ± 1.7 × 10−5 μg/mL (0.70 ± 0.37 pM). The microfluidic biosensor can also be used for determination of the apparent dissociation constant (Kd) between SLP and anti-SLP, which was measured to be 4.8 ± 0.9 × 10-9 M.

Comparative study of prostate cancer biophysical and migratory characteristics via iterative mechanoelectrical properties (iMEP) and standard migration assays

This study reveals a new microfluidic biosensor consisting of a multi-constriction microfluidic device with embedded electrodes for measuring the biophysical attributes of single cells. The biosensing platform called the iterative mechano-electrical properties (iMEP) analyzer captures electronic records of biomechanical and bioelectrical properties of cells. The iMEP assay is used in conjunction with standard migration assays, such as chemotaxis-based Boyden chamber and scratch wound healing assays, to evaluate the migratory behavior and biophysical properties of prostate cancer cells. The three cell lines evaluated in the study each represent a stage in the standard progression of prostate cancer, while the fourth cell line serves as a normal/healthy counterpart. Neither the scratch assay nor the chemotaxis assay could fully differentiate the four cell lines. Furthermore, there was not a direct correlation between wound healing rate or the migratory rate with the cells’ metastatic potential. However, the iMEP assay, through its multiparametric dataset, could distinguish between all four cell line populations with p-value < 0.05. Further studies are needed to determine if iMEP signatures can be used for a wider range of human cells to assess the tumorigenicity of a cell population or the metastatic potential of cancer cells.

Ultrasensitive electrochemiluminescence detection of p53 gene by a novel cloth-based microfluidic biosensor with luminol-gold nanoparticles and hybridization chain reaction amplification

A dual-signal-amplified cloth-based ECL biosensor has been firstly explored to detect p53 gene, where the luminol functionalized gold nanoparticles (Lu-AuNPs) are used for probe labels and signal amplification, and the hybridization chain reaction (HCR) is further applied to increase the sensitivity of ECL detection. Carbon ink/solid wax-based screen-printing is utilized to manufacture the cloth-based carbon electrodes and sample/auxiliary chambers to construct a collapsible three-dimensional (3-D) cloth-based device. Profiting from the 3-D device feature, it is easy to prepare the sensing interface modified by multi-walled carbon nanotubes. When the target DNA, signal nanoprobes and hydrogen peroxide (H2O2) are successively dropped onto the sensing interface, the Lu/H2O2-based ECL can be operated well. Under optimal conditions, the p53 gene can be desirably detected, with a linear range of 0.0001–1000 pM and detection limit of 0.02 fM. The biosensor has a wider linear range, and relatively high sensitivity, selectivity, stability and reproducibility, possibly because of the excellent constitution of signal nanoprobes and HCR products. Peculiarly, the biosensor has the capacity for p53 gene detection in the complex cellular homogenate samples, and discrimination of one-, two- and even three-base-mismatched p53 DNA sequences. So, it may supply a hopeful method in applications of gene diagnostics.

Numerical simulation of a microfluidic biosensor for C-reactive protein detection into a microchannel with considering electrothermal effect

The performance enhancement of an electrothermal effect-based biosensor is studied numerically in this research. Indeed, the study of a microfluidic biosensor is used to detect the C-reactive protein into a microchannel with considering electrothermal effect. In the presence of the electrical field, the produced Joule effect gives rise to a temperature gradient and causes homogeneities of electric permittivity and conductivity allowing the generation of flow motions. To improve the rate of reactant ransport via an on-surface-reaction in the microchannel, various pairs of electrodes under Alternating current (AC) are used and the effects of different arrangements and positions on flow structure, temperature and concentration fields, were investigated. In addition, the comparison with earliest results shows a good agreement.

A Rapid and Sensitive Salmonella Biosensor Based on Viscoelastic Inertial Microfluidics.

Salmonella is a main cause of foodborne illnesses and rapid screening of Salmonella is the key to prevent Salmonella outbreaks, however available detection methods either require a long time, or need complex pretreatment, or have low sensitivity. In this study, a microfluidic biosensor was developed for Salmonella detection using viscoelastic inertial microfluidics for separating magnetic bacteria from unbound magnetic nanoparticles (MNPs) and enzyme catalytic colorimetry for amplifying biological signals. The polyclonal antibodies and horseradish peroxidase (HRP) modified MNPs were first used to specifically capture Salmonella to form magnetic HRP-bacteria. Both magnetic HRP-bacteria and unbound MNPs were magnetically separated from background and resuspended in viscoelastic polyvinylpyrrolidone solution as sample flow. When sample flow was injected with polyvinylpyrrolidone sheath flow into a T-shaped microchannel, larger-sized magnetic HRP-bacteria could penetrate the sample flow, however smaller-sized MNPs remained in the sample flow due to weaker inertial lift force and elastic lift force, resulting in continuous-flow separation of magnetic HRP-bacteria. Finally, magnetic HRP-bacteria were collected and concentrated to catalyze tetramethyl benzidine, and absorbance was measured to determine the bacteria. This biosensor was able to detect Salmonella as low as 30 CFU/mL in 1 h and featured the advantages of shorter time due to a one-step immunoreaction, easier extension due to only one antibody and one label, and lower cost due to less expensive materials.

Bioactive microfluidic paper device for pesticide determination in waters

This work presents a new optical microfluidic paper biosensor for the detection of organophosphate pesticides and carbamate pesticides. The assay strip is composed of a paper support (1 × 17.6 mm) onto which acetylcholine esterase (AChE) and acetylcholine chloride (AChCl) are deposited, in such a way that there is a small hole between them that ensures that they only come into contact in the reaction zone when they are carried by a solution of the sample by lateral flow to the reaction zone containing bromocresol purple (BCP) as the pH indicator, immobilized by sol-gel. The sensor operates at room temperature and the rate of the inhibited reaction serves as an analytical signal, which is measured using a camera by quantifying the appropriate colour coordinate. Calibration curves were obtained for chlorpyrifos and carbaryl, with a useful concentration range from 0.24 to 20 μg L−1 for carbaryl and from 2.00 to 45 μg L−1 for chlorpyrifos. The detection limits were 0.24 and 2.00 μg L−1, respectively, and with reproducibility around 4.2–5.5%. The method was applied to the determination of pesticides in different water samples, with no sample preparation.

Engineered Microneedles for Interstitial Fluid Cell‐Free DNA Capture and Sensing Using Iontophoretic Dual‐Extraction Wearable Patch

AbstractInterstitial fluid (ISF), as an emerging source of biomarkers, is unmistakably significant for disease diagnosis. Microneedles (MNs) provide a minimally invasive approach for extracting the desired molecules from ISF. However, existing MNs are limited by their capture efficiency and sensitivity, which impedes early disease diagnosis. Herein, an engineered wearable epidermal system is presented with a combination of reverse iontophoresis and MNs for rapid capture and sensing of Epstein‐Barr virus cell‐free DNA (an important biomarker of nasopharyngeal carcinoma). Owing to a dual‐extraction effect demonstrated by reverse iontophoresis and MNs, the engineered wearable platform successfully isolates the cell‐free DNA target from ISF within 10 min, with a threshold of 5 copies per µL and a maximum capture efficiency of 95.4%. The captured cell‐free DNA is also directly used in a recombinase polymerase amplification electrochemical microfluidic biosensor with a detection limit of 1.1 copies per µL (or a single copy). The experimental data from immunodeficient mouse models rationalizes the feasibility and practicality of the wearable system. Collectively, the developed method opens an innovative route for minimally invasive sampling of ISF for cell‐free DNA‐related cancer screening and prognosis.

Biomaterial Embedding Process for Ceramic-Polymer Microfluidic Sensors.

One of the major issues in microfluidic biosensors is biolayer deposition. Typical manufacturing processes, such as firing of ceramics and anodic bonding of silicon and glass, involve exposure to high temperatures, which any biomaterial is very vulnerable to. Therefore, current methods are based on deposition from liquid, for example, chemical bath deposition (CBD) and electrodeposition (ED). However, such approaches are not suitable for many biomaterials. This problem was partially resolved by introduction of ceramic–polymer bonding using plasma treatment. This method introduces an approximately 15-min-long window for biomodification between plasma activation and sealing the system with a polymer cap. Unfortunately, some biochemical processes are rather slow, and this time is not sufficient for the proper attachment of a biomaterial to the surface. Therefore, a novel method, based on plasma activation after biomodification, is introduced. Crucially, the discharge occurs selectively; otherwise, it would etch the biomaterial. Difficulties in manufacturing ceramic biosensors could be overcome by selective surface modification using plasma treatment and bonding to polymer. The area of plasma modification was investigated through contact-angle measurements and Fourier-transform infrared (FTIR) analyses. A sample structure was manufactured in order to prove the concept. The results show that the method is viable.

A Microfluidic Biosensor Based on Magnetic Nanoparticle Separation, Quantum Dots Labeling and MnO2 Nanoflower Amplification for Rapid and Sensitive Detection of Salmonella Typhimurium.

Screening of foodborne pathogens is an effective way to prevent microbial food poisoning. A microfluidic biosensor was developed for rapid and sensitive detection of Salmonella Typhimurium using quantum dots (QDs) as fluorescent probes for sensor readout and manganese dioxide nanoflowers (MnO2 NFs) and as QDs nanocarriers for signal amplification. Prior to testing, amino-modified MnO2 nanoflowers (MnO2-NH2 NFs) were conjugated with carboxyl-modified QDs through EDC/NHSS method to form MnO2-QD NFs, and MnO2-QD NFs were functionalized with polyclonal antibodies (pAbs) to form MnO2-QD-pAb NFs. First, the mixture of target Salmonella Typhimurium cells and magnetic nanoparticles (MNPs) modified with monoclonal antibodies (mAbs) was injected with MnO2-QD-pAb NFs into a microfluidic chip to form MNP-bacteria-QD-MnO2 complexes. Then, glutathione (GSH) was injected to dissolve MnO2 on the complexes into Mn2+, resulting in the release of QDs. Finally, fluorescent intensity of the released QDs was measured using the fluorescent detector to determine the amount of Salmonella. A linear relationship between fluorescent intensity and bacterial concentration from 1.0 × 102 to 1.0 × 107 CFU/mL was found with a low detection limit of 43 CFU/mL and mean recovery of 99.7% for Salmonella in spiked chicken meats, indicating the feasibility of this biosensor for practical applications.

An Acid-Responsive Microfluidic Salmonella Biosensor Using Curcumin as Signal Reporter and ZnO-Capped Mesoporous Silica Nanoparticles for Signal Amplification

An acid-responsive microfluidic biosensor using curcumin (CUR) as signal reporter and ZnO-capped mesoporous silica nanoparticles (MSNs) for signal amplification was developed for sensitive and rapid detection of Salmonella in colorimetric and fluorescent modal. The MSNs were first incubated with CUR to obtain MSN@CUR nanoparticles (MC NPs). The MC NPs were then capped with ZnO nanoparticles to form MSN@CUR@ZnO nanoparticles (MCZ NPs) to prevent CUR from premature release, followed by modification with polyclonal antibodies (pAbs) against Salmonella Typhimurium (S. Typhimurium) to obtain immune MSN@CUR@ZnO@pAbs nanoparticles (MCZP NPs). The immune magnetic nanoparticles (MNPs), the S. Typhimurium cells and the MCZP NPs were conjugated in the microfluidic chip with a Koch fractal mixing channel to form MNP-bacteria-MCZP complexes. Finally, the acetic acid (HAc) was first time introduced to release CUR from the complexes, and both the color and fluorescence changes were measured to determine the concentration of Salmonella. This proposed biosensor was able to quantitatively detect S. Typhimurium ranging from 102 to 107 CFU/mL in 1.5 h and the lower detection limits were calculated to be 63 CFU/mL for colorimetric measurement and 40 CFU/mL for fluorescent measurement, respectively. The mean recovery was about 104 % for Salmonella in the spiked chicken samples.

Lab-On-Chip, Internet of Things, Analytics and Health Care 4.0: A synergistic future forward

In this era of the internet with the latest technology and rapid communication, the various fields of processes and business are led to the road of automation in recent days. The major part of this automation is that they help in the cost reduction with increased safety measures and also results in absolute portability of the equipment. To indulge in continuous processing, and also to identify the best technology, the analytical methods must be directly involved in decision making. This revolution in analytical methods is named as Analytics 4.0, which has a significant role in healthcare and diagnostics. The self-assessment and decentralization of the data acquiring can be motivated with the help of the microfluidic biosensors and biochip. It also helps in the immediate access of the information without resulting in delay. Diagnostic 4.0 helps in storing the patient details as cloud files providing high security and also analyze them through the deep learning algorithms. It also helps in constant monitoring on a broad scale and reliable assessment through monitoring apps that can shift the health care efficiency to the next notch. Hence the evolution of the healthcare sector has multiple generations listed with numerous advances which stand as the boon in this era.

Capillary-assisted microfluidic biosensing platform captures single cell secretion dynamics in nanoliter compartments

Cancer cells continuously secrete inflammatory biomolecules which play significant roles in disease progression and tumor metastasis toward secondary sites. Despite recent efforts to capture cancer cells' intercellular secretion heterogeneity using microfluidics, the challenges in operation of these systems as well as the complexity of designing a biosensing assay for long-term and real-time measurement of single cell secretions have become grand research barriers. Here, we present a new capillary-based microfluidic biosensing approach to easily and reliably capture ~500 single cells inside isolated dead-end nanoliter compartments using simple pipette injection, and quantify their individual secretion dynamics at the single cell resolution over a long period of culture (~16 h). We first present a detailed investigation of the fluid mechanics underlying the formation of nanoliter compartments in the microfluidic system. Based on the measurement of single cell capture efficiency, we employ a one-step FRET-based biosensor which monitors the single cancer cells' protease activity. The sensor reports the fluorescent signal as a product of amino acid chain cleavage and reduction in its quenching capability. Using the single cell protease secretion data, we identified modes of cell secretion dynamics in our cell sample. While most of the cells had low secretion levels, two other smaller and more aggressive secretion dynamics were cells with secretion modes that include sharp spikes or slow but progressive trend. The method presented here overcomes the difficulties associated with performing single cell secretion assays, enabling a feasible and reliable technique for high throughput measurement of metabolic activities in cancer cells.

Novel Nanoplasmonic-Structure-Based Integrated Microfluidic Biosensors for Label-Free in Situ Immune Functional Analysis: A review of recent progress.

The study of immune functional responses is essential to understanding the central role of the immune system in providing immunological host defense and its intercommunication with other systems. The recent development of integrated microfluidic cytokine biosensors has established a new paradigm to identify, isolate, and study immune cell subtypes, cell functions, and intercellular communications that constitute those responses.

A comparative study on EpCAM antibody immobilization on gold surfaces and microfluidic channels for the detection of circulating tumor cells

Detection of circulating tumor cells (CTCs) from the bloodstream holds great importance to diagnose cancer at early stages. However, CTCs being extremely rare in blood makes them difficult to reach. In this paper, we introduced different surface modification techniques for the enrichment and detection of MCF-7 in microfluidic biosensor applications using gold surface and EpCAM antibody. Mainly, two different mechanisms were employed to immobilize the antibodies; covalent bonding and bioaffinity interaction. Self-assembled monolayers (SAMs) formed on the gold surfaces were treated further for the immobilization of the antibody. The bioaffinity-based studies were performed with streptavidin and biotinylated EpCAM over the SAM coated surfaces. The cell attachment events were monitored using fluorescent microscope. Comparisons were made considering the length and functional end of alkanethiols and the positioning of the antibody. Then, these methods were integrated into a microfluidic channel system. Surface characterizations were performed with X-ray Photoelectron Spectroscopy, Atomic Force Microscopy, and contact angle measurements. The selectivity studies were carried out with EpCAM negative K562 leukaemia cell lines and the experiments were repeated for different types of surfaces, such as glass and polymer. Studies showed that long (n>10) and aromatic ring containing alkanethiols lead to better cell capture events compared to shorter ones. Results obtained from the comparisons are of importance for the gold surface-based microfluidic biosensor designs aimed for CTC detection.

Shape design for stabilizing microparticles in inertial microfluidic flows

Design of microparticles which stabilize at the centerline of a channel flow when part of a dilute suspension is examined numerically for moderate Reynolds numbers ($10 \le Re \le 80$). Stability metrics for particles with arbitrary shapes are formulated based on linear-stability theory. Particle shape is parametrized by a compact, Non-Uniform Rational B-Spline (NURBS)-based representation. Shape-design is posed as an optimization problem and solved using adaptive Bayesian optimization. We focus on designing particles for maximal stability at the channel-centerline robust to perturbations. Our results indicate that centerline-focusing particles are families of characteristic "fish"/"bottle"/"dumbbell"-like shapes, exhibiting fore-aft asymmetry. A parametric exploration is then performed to identify stable particle-designs at different k's (particle chord-to-channel width ratio) and Re's ($0.1 \le k \le 0.4, 10 \le Re \le 80$). Particles at high-k's and Re's are highly stabilized when compared to those at low-k's and Re's. A comparison of the modified dumbbell designs from the current framework also shows better performance to perturbations in Fluid-Structure Interaction (FSI) when compared to the rod-disk model reported previously (Uspal & Doyle 2014) for low-Re Hele-Shaw flow. We identify basins of attraction around the centerline, which span larger release-angle-ranges and lateral locations (tending to the channel width) for narrower channels, which effectively standardizes the notion of global focusing in such configurations using the current stability-paradigm. The present framework is illustrated for 2D cases and is potentially generalizable to stability in 3D flow-fields. The current formulation is also agnostic to Re and particle/channel geometry which indicates substantial potential for integration with imaging flow-cytometry tools and microfluidic biosensing-assays.

A highly efficient preconcentration route for rapid and sensitive detection of endotoxin based on an electrochemical biosensor.

An impedimetric aptasensor for the detection of endotoxin in a microfluidic chip was proposed, in which the Apt/AuNPs/SPCE sensing surface was fabricated in a screen-printed electrode with good biological activity and stability. The quantitative detection of endotoxin was accomplished by electrochemical impedance spectroscopy (EIS) measurement before and after exposing to samples. The impedance biosensor offers an ultrasensitive and selective detection of endotoxin down to 500 pg mL-1 with a wide linear range from 500 pg mL-1 to 200 ng mL-1. According to the Langmuir isotherm model, the interactions between the target molecules and the sensing surface had been analyzed and strong binding was concluded. Compared to the traditional static incubation methods, the microfluidic biosensor realizes the enrichment of endotoxin owing to the confined space and continuous flow nature, so that the lowest detection concentration is reduced from 5 ng mL-1 to 500 pg mL-1, which is much lower than the existing technology, and the total assay time is shortened from 1.0 h to 0.5 h. The proposed microfluidic impedance biosensor provides a new strategy for the design of an aptasensor to realize the rapid detection of target biomolecules with high sensitivity and it can be integrated into wearable medical devices due to its flexible properties.