Microfluidic-based biosensors toward point-of-care detection of nucleic acids and proteins

Abstract

This article reviews state-of-the-art microfluidic biosensors of nucleic acids and proteins for point-of-care (POC) diagnostics. Microfluidics is capable of analyzing small sample volumes (10^-9-10^-18 l) and minimizing costly reagent consumption as well as automating sample preparation and reducing processing time. The merger of microfluidics and advanced biosensor technologies offers new promises for POC diagnostics, including high-throughput analysis, portability and disposability. However, this merger also imposes technological challenges on biosensors, such as high sensitivity and selectivity requirements with sample volumes orders of magnitude smaller than those of conventional practices, false response errors due to non-specific adsorption, and integrability with other necessary modules. There have been many prior review articles on microfluidic-based biosensors, and this review focuses on the recent progress in last 5 years. Herein, we review general technologies of DNA and protein biosensors. Then, recent advances on the coupling of the biosensors to microfluidics are highlighted. Finally, we discuss the key challenges and potential solutions for transforming microfluidic biosensors into POC diagnostic applications.

Keywords: Biosensor; DNA; Microfluidics; Point-of-care detection; Protein.

Fig. 1

Examples of microfluidic biosensors using semiconductor nanowires. a Prototype nanowire sensor biochip with integrated microfluidic sample delivery (Patolsky et al. 2006). b The experiment by Stern et al. demonstrated that nanowires can be used to detect DNA hybridization. In panel A, a cross section schematic of the device shows the immobilized DNA on top of the nanowire with its source and drain contacts. Panels B and C show the source drain current change for two devices that have been functionalized with specific probe DNA strands upon addition of target DNA. The device in Panel B has been functionalized with probe 1, the one in C with probe 2, respectively. If 10 pM of the respective complementary target is added to the solution, the drain current increases (bottom trace), while the current stays constant if a non-complementary strand is introduced (top trace). The DNA sequences used were DNA-T(1): 5′-CCT GCA GTG ACG CAG TGGCG-3′; DNA-T(2): 5′-AAG GTG GAA AAT GTA ATC TA-3′;DNA-P(1): 5′-CGC CAC TGC GTC ACT GCA GG-3′; DNAP(2): 5′-TAG ATT ACA TTT TCC ACC TT-3′. In order to observe the drain current change, the solution has to be of a low ionic concentration, 5 mM, that the Debye length is larger than the length of the DNA fragment (Stern et al. 2007b)

Fig. 2

a A Schematic diagram for measurements of electrical characteristics of a genetic field effect transistor (FET). The FET can be integrated into a microfluidic channel to allow in situ detection. B Electrical signal of immobilization of oligonucleotide probes, hybridization with target DNA on the FET (Sakata et al. 2004). b Direct DNA sequence readout using a natural nanopore (α-Haemolysin). The current versus time traces on the right show that a poly(dC) strand exhibits a different current signature when compared to a poly(dA) strand, allowing to draw a conclusions of which bases are present. By modifying the lumen of the a-HL pore the current signal can be improved (Stoddart et al. ; Copyright (2009) National Academy of Sciences, U.S.A)

Fig. 3

Example of a micro-ELISA in microwells fabricated using SU-8 epoxy patterned on silicon. An IgG immunoassay has been performed inside of these microwells. a A schematic of the microwell chip, b a fluorescence image (top view) of the microwell plate, c a scanning electron micrograph of a single well and d a fluorescence microscopy image of the immunoassay on a part of the chip (C-reactive protein, Cy5 fluorescent dyes). The detection limit was reported to be 30 ng/ml and the assay time was 4 h. The microfluidic approach helps to reduce the solution volume, consequently reducing assay time (Blagoi et al. 2008)

Fig. 4

a A microbead-based immunoassay, performed inside of a microfluidic chip. Polystyrene beads are used to immobilize antibodies. The dam structure inside the channel prevents the beads from entering the measurement site (Kakuta et al. 2006). b ImmuChip, implementing a miniaturized enzyme-linked immunoassay on a microfluidic chip, thereby reducing the sample volume. The integrated gold working electrode and Ag/AgCl reference electrodes allow an electrochemical readout of the immunoreaction (Hoegger et al. 2007)

Fig. 5

Microfluidic immunosensor based on the separation of magnetic microbeads, using impedance spectroscopy for detection. a The assembly of the microfluidic chip, consisting of a microfluidic bottom layer containing 7 parallel channels, an interdigitated array (IDA) middle layer providing electrical contact to the microchannels and a top layer containing a patterned permalloy to allow magnetic bead separation. All parts were fabricated using injection molding and bonded using an UV-curable adhesive. b The complete assembly with a magnified view of the electrode array and the permalloy magnetic bead separator (Do and Ahn 2008)

Fig. 6

Parallel microchannel device for the detection and determination of pollen. Due to the different surface charge of pollen and polystyrene test particles, the Coulter signature is different between the two types of particles, despite of similar sizes (Zhe et al. 2007)

Fig. 7

a A custom-made microfluidic device to demonstrate the Vroman effect-based protein biosensor. b A schematic of operating principle. (1) IgG is injected from the inlet 1 to cover both surfaces, (2) washing process to remove unbound IgG, (3) fibrinogen flows from inlet 2 and displaces the pre-adsorbed IgG on one surface, (4) washing process to remove any residue on the surface, (5) a mixture of albumin, haptoglobin and Tg flows from inlet 1, (6) only Tg displaces IgG in channel 1 while any of proteins does not displace fibrinogen in channel 2. c SPR sensorgram of the displacement event; Tg detection of two engineered surfaces, pre-adsorbed by IgG and fibrinogen. d Normalized close-up SPR sensorgram after the Tg injection, e final angle changes (%) on both surfaces (angle change/previous angle value × 100). Each has selectivity to a specific protein to be detected (Choi and Chae 2009b)

Fig. 8

μPADs for analysis of glucose and protein in urine. a Patterned paper after distributing 5 μl of red ink to show the integrity of the hydrophilic channel. b Complete μPADs after spotting the reagents. c Positive assays for glucose and protein using 5 μl of a solution that contained glucose and BSA in an artificial urine solution. d Results of paper-based glucose and protein assays using a range of concentrations of glucose and BSA in artificial urine (Martinez et al. 2010)

Fig. 9

a An integrated microfluidic platform fabricated by multilayer soft lithography (Grover et al. 2006). b Schematic of the digital microfluidic platform (Lienemann et al. 2006). c Formation of water-in-silicone oil droplets with an embedded circular orifice (Yobas et al. 2006). d A microelectrode array device (Krishnan et al. ; Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission)