Toward a solid-phase nucleic acid hybridization assay within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer

The optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD–oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel for re-use of the microfluidic chip.     

New advances in electrochemical biosensors for the detection of toxins: Nanomaterials,magnetic beads and microfluidics systems. A review

The use of nanotechnology in bioanalytical devices has special advantages in the detection of toxins of interest in food safety and environmental applications. The low levels to be detected and the small size of toxins justify the increasing number of publications dealing with electrochemical biosensors, due to their high sensitivity and design versatility. The incorporation of nanomaterials in their development has been exploited to further increase their sensitivity, providing simple and fast devices, with multiplexed capabilities. This paper gives an overview of the electrochemical biosensors that have incorporated carbon and metal nanomaterials in their configurations for the detection of toxins. Biosensing systems based on magnetic beads or integrated into microfluidics systems have also been considered because of their contribution to the development of compact analytical devices. The roles of these materials, the methods used for their incorporation in the biosensor configurations as well as the advantages they provide to the analyses are summarised.     

Detection of E. coli using a microfluidics-based antibody biochip detection system

This work demonstrates the detection of E. coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays. The biochip features a 4 × 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform. The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection. Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays. Received: 13 October 2000 / Revised: 13 November 2000 / Accepted: 13 November 2000     

Detection of E. coli using a microfluidics-based antibody biochip detection system

This work demonstrates the detection of E. coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays. The biochip features a 4 x 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform. The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection. Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays.     

Trends in DNA biosensors

Biosensors have witnessed an escalating interest nowadays, both in the research and commercial fields. Deoxyribonucleic acid (DNA) biosensors (genosensors) have been exploited for their inherent physico-chemical stability and suitability to discriminate different organism strains. The main principle of detection among genosensors relies on specific DNA hybridization, directly on the surface of a physical transducer. This review covers the main DNA immobilization techniques reported so far, new micro- and nanotechnological platforms for biosensing and the transduction mechanisms in genosensors. Clinical applications, in particular, demand large-scale and decentralized DNA testing. New schemes for DNA diagnosis include DNA chips and microfluidics, which couples DNA detection with sample pretreatment under in vivo-like hybridization conditions. Higher sensitivity and specificity may arise from nanoengineered structures, like carbon nanotubes (CNTs) and DNA/protein conjugates. A new platform for universal DNA biosensing is also presented, and its implications for the future of molecular diagnosis are argued.     

A biosensor based on graphite epoxy composite electrode for aspartame and ethanol detection

A gelatin membrane with carboxyl esterase and alcohol oxidase was subsequently integrated onto the surface of a graphite epoxy composite electrode (GECE). The developed biosensors showed linearity in the range of 2.5–400 μM for aspartame and 2.5–25 μM for ethanol with response times of 170 and 70 s for each analyte, respectively. The resulting bienzyme biosensor was used for aspartame detection in diet coke samples and ethanol detection in beer and wine samples. From the obtained results, it can be concluded that the developed biosensor is a selective, practical and economic tool for aspartame and ethanol detection in real samples.     

A novel microbial biosensor based on cells of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> for the selective determination of 1,3-propanediol in the presence of glycerol and its application to bioprocess monitoring

Novel and selective microbial amperometric biosensors that use Gluconobacter oxydans cells to monitor the bacterial bioconversion of glycerol (Gly) to 1,3-propanediol (1,3-PD) are described. Two different mediators, ferricyanide and flexible polyvinylimidazole osmium functionalized polymer (Os-polymer), were employed to prepare two different microbial biosensors, both of which gave high detection performance. The good operational stabilities of both types of biosensor were underlined by the ability to detect 1,3-PD throughout 140 h of continuous operation. Both microbial biosensor systems showed excellent selectivity for 1,3-PD in the presence of a high excess of glycerol [selectivity ratios (1,3-PD/Gly) of 118 or 245 for the ferricyanide and Os-polymer systems, respectively]. Further, the robustness of each microbial biosensor was highlighted by the high reliability of 1,3-PD detection achieved (average RSD of standards <2%, and well below 4% for samples). The biosensor implementing the Os-polymer mediator exhibited high selectivity towards 1,3-PD detection and allowed moderate sample throughput (up to 12 h⁻¹) when integrated into a flow system. This system was used to monitor the concentration of 1,3-PD during a real bioprocess. Results from biosensor assays of 1,3-PD in bioprocess samples taken throughout the fermentation were in a very good agreement with results obtained from reference HPLC assays (R ² = 0.999).     

Research progress on pesticide residue detection based on microfluidic technology

The problem of pesticide residue contamination has attracted widespread attention and poses a risk to human health. The current traditional pesticide residue detection methods have difficulty meeting rapid and diverse field screening requirements. Microfluidic technology integrates functions from sample preparation to detection, showing great potential for quick and accurate high-throughput detection of pesticide residues. This paper reviews the latest research progress on microfluidic technology for pesticide residue detection. First, the commonly used microfluidic materials are summarized, including silicon, glass, paper, polydimethylsiloxane, and polymethyl methacrylate. We evaluated their advantages and disadvantages in pesticide residue detection applications. Second, the current pesticide residue detection technology based on microfluidics and its application to real samples are summarized. Finally, we discuss this technology's present challenges and future research directions. This study is expected to provide a reference for the future development of microfluidic technology for pesticide residue detection.     

Chemical surface modifications for the development of silicon-based label-free integrated optical (IO) biosensors: A review

Increasing interest has been paid to label-free biosensors in recent years. Among them, refractive index (RI) optical biosensors enable high density and the chip-scale integration of optical components. This makes them more appealing to help develop lab-on-a-chip devices. Today, many RI integrated optical (IO) devices are made using silicon-based materials. A key issue in their development is the biofunctionalization of sensing surfaces because they provide a specific, sensitive response to the analyte of interest. This review critically discusses the biofunctionalization procedures, assay formats and characterization techniques employed in setting up IO biosensors. In addition, it provides the most relevant results obtained from using these devices for real sample biosensing. Finally, an overview of the most promising future developments in the fields of chemical surface modification and capture agent attachment for IO biosensors follows.     

Droplet microfluidics based microseparation systems

Lab on a chip (LOC) technology is a promising miniaturization approach. The feature that it significantly reduced sample consumption makes great sense in analytical and bioanalytical chemistry. Since the start of LOC technology, much attention has been focused on continuous flow microfluidic systems. At the turn of the century, droplet microfluidics, which was also termed segmented flow microfluidics, was introduced. Droplet microfluidics employs two immiscible phases to form discrete droplets, which are ideal vessels with confined volume, restricted dispersion, limited cross-contamination, and high surface area. Due to these unique features, droplet microfluidics proves to be a versatile tool in microscale sample handling. This article reviews the utility of droplet microfluidics in microanalytical systems with an emphasize on separation science, including sample encapsulation at ultra-small volume, compartmentalization of separation bands, isolation of droplet contents, and related detection techniques.     

微流控芯片系统在循环肿瘤细胞分离检测中的应用进展

循环肿瘤细胞(CTCs)的分离分析一直是肿瘤相关研究中的热点方向,作为液体活检的重要标志物之一,其在外周血中的含量与癌症病发状况密切相关.然而人体血液中CTCs的含量非常低,通常来说仅有0～10个/mL,因此在开展临床血液样本中CTCs的检测前,往往需要对样本进行前处理,以实现CTCs的分离和富集.微流控芯片技术凭借样...     

Electrochemical biosensors based on Ti3C2Tx MXene: future perspectives for on-site analysis

MXenes are recently developed two-dimensional layered materials composed of early transition metal carbides and/or nitrides that provide unique characteristics for biosensor applications. This review presents the recent progress made on the usage and applications of MXenes in the field of electrochemical biosensors, including microfluidic biosensors and wearable microfluidic biosensors, and highlights the challenges with possible solutions and future needs. The multilayered configuration and high conductivity make these materials as an immobilization matrix for the biomolecule immobilization with activity retention and to be explored in the fabrication of electrochemical sensors, respectively. First, how the MXene nanocomposite as an electrode modifier affects the sensing performance of the electrochemical biosensors based on enzymes, aptamer/DNA, and immunoassays is well described. Second, recent developments in MXene nanocomposites as wearable biosensing platforms for the biomolecule detection are highlighted. This review pointed out the future concerns and directions for the use of MXene nanocomposites to fabricate advanced electrochemical biosensors with high sensitivity and selectivity. Specifically, possibilities for developing microfluidic electrochemical sensors and wearable electrochemical microfluidic sensors with integrated biomolecule detection are emphasized.     

Patterning chemical stimulation of reconstructed neuronal networks

A spatially resolved delivery of substances integrated with cell culture substrates shows promise for application in pharmacological assays, bioanalytical studies on cell signaling pathways and cell-based biosensors, where control over the extracellular biochemical environment with a cellular resolution is desirable. In this work, we studied a biohybrid system where rat embryonic cortical neuronal networks are reconstructed on microstructured silicon chips and interfaced to microfluidics. The design of cell-cell and cell-medium interactions in confined geometries is presented. We developed an aligned microcontact printing technique (AμCP) for poly(lysine)-extracellular matrix proteins on microstructured chips, which allows a high degree of geometrical control over the network architecture and alignment of the neuronal network with the microfluidic features of a substrate. Spatially resolved on-chip delivery of compounds with a cellular resolution is demonstrated by chemical stimulation of patterned rat cortical neurons within a network with a number of solutions of excitatory neurotransmitter glutamate delivered via microfluidics. The combination of the system described with a patch-clamp technique allowed both modulation of the biochemical environment on a cellular level and the monitoring of electrophysiological properties in the reconstructed rat embryonic cortical networks changed by this microenvironment.     

Nanophotonic lab-on-a-chip platforms including novel bimodal interferometers, microfluidics and grating couplers

One of the main limitations for achieving truly lab-on-a-chip (LOC) devices for point-of-care diagnosis is the incorporation of the "on-chip" detection. Indeed, most of the state-of-the-art LOC devices usually require complex read-out instrumentation, losing the main advantages of portability and simplicity. In this context, we present our last advances towards the achievement of a portable and label-free LOC platform with highly sensitive "on-chip" detection by using nanophotonic biosensors. Bimodal waveguide interferometers fabricated by standard silicon processes have been integrated with sub-micronic grating couplers for efficient light in-coupling, showing a phase resolution of 6.6 × 10(-4)× 2π rad and a limit of detection of 3.3 × 10(-7) refractive index unit (RIU) in bulk. A 3D network of SU-8 polymer microfluidics monolithically assembled at the wafer-level was included, ensuring perfect sealing and compact packaging. To overcome some of the drawbacks inherent to interferometric read-outs, a novel all-optical wavelength modulation system has been implemented, providing a linear response and a direct read-out of the phase variation. Sensitivity, specificity and reproducibility of the wavelength modulated BiMW sensor has been demonstrated through the label-free immunodetection of the human hormone hTSH at picomolar level using a reliable biofunctionalization process.     

Recent trends in nanomaterial-based microanalytical systems for the speciation of trace elements: A critical review

Trace element speciation in biomedical and environmental science has gained increasing attention over the past decade as researchers have begun to realize its importance in toxicological studies. Several nanomaterials, including titanium dioxide nanoparticles (nano-TiO₂), carbon nanotubes (CNTs), and magnetic nanoparticles (MNPs), have been used as sorbents to separate and preconcentrate trace element species prior to detection through mass spectrometry or optical spectroscopy. Recently, these nanomaterial-based speciation techniques have been integrated with microfluidics to minimize sample and reagent consumption and simplify analyses. This review provides a critical look into the present state and recent applications of nanomaterial-based microanalytical systems in the speciation of trace elements. The adsorption and preconcentration efficiencies, sample volume requirements, and detection limits of these nanomaterial-based speciation techniques are detailed, and their applications in environmental and biological analyses are discussed. Current perspectives and future trends into the increasing use of nanomaterial-based microfluidic techniques for trace element speciation are highlighted.     

Active Junctions to Improve Sensitivity and Detection Limit of a Microdevice Based on Coulometry Coupled with Silver Metallization

For achieving miniaturization and coupling with microfluidics, electrochemical devices are advantageous. In particular, coulometry is an effective tool to analyze components in solutions of very small volumes. The sensitivity and detection limit of coulometry can be improved remarkably by converting an analyte into Ag on the same Pt electrode separated in two flow channels and measuring the amount of Ag using coulometry. We demonstrate that the cathodic and anodic potentials of both parts of the Pt electrode can be shifted and reactions can be accelerated by replacing the liquid junction connecting the two flow channels by a metal wire with redox active ends. This in turn improves the sensitivity and detection limit of the device. This effect can also be observed by directly applying a voltage to the solutions in the flow channels. The achieved detection limit for hydrogen peroxide (H₂O₂) was 2.4 nM.     

Metal-polymer nanocomposites for integrated microfluidic separations and surface enhanced Raman spectroscopic detection

The widespread development of microfluidics (microfluidics) has allowed the extension of efficient separations, fluid handling, and hyphenation with many detection modes to a small, portable, highly controllable physico-chemical platform. Surface enhanced Raman spectroscopy (SERS) offers the powerful advantage of obtaining vibrational spectroscopic information about analytes in an aqueous matrix with negligible background. The mating of electrophoretic separations with vibrational spectroscopy on a microfluidic device will allow the chromatographic efficiency of capillary electrophoresis (CE) with the unequivocal analyte "fingerprinting" capability of detailed structural information. By utilizing SERS as a means of detection, this work promises to yield redress for the hindrances of electrophoretic separations, including uncertainty in analyte band identification due to changing migration times as well as compromised detection sensitivity for non-fluorescent analytes. Our work represents the first steps toward developing CE-SERS on a microfluidic platform with a region of novel metal-pliable polymer nanocomposite SERS substrate fabricated directly into the device. The device fabrication material has been extensively employed by the microfluidics community for over five years. SERS detection can be achieved in real time or after the separations, with on-column laser-induced fluorescence employed as a secondary detection mode used for confirmation of efficiencies and band locations.     

A 96-well microplate incorporating a replica molded microfluidic network integrated with photonic crystal biosensors for high throughput kinetic biomolecular interaction analysis

A nanoreplica molding process has been used to produce polymer microfluidic channels, with integrated label-free photonic crystal biosensors as the bottom surface of the channels. Multiple flow channels are gathered in parallel so that an imaging detection instrument may simultaneously monitor the binding kinetics of many biomolecular interactions. In this work, the flow channel pattern has been adapted to a 96-well microplate format in which, for each 12-element row of the microplate, a single well serves as a common access port for 11 flow channels that are connected to separate microplate wells. Application of pneumatic pressure or suction to the common well serves to drive forward or backward flow to the channels. The system is demonstrated by measuring the kinetic binding interaction of protein A with IgG molecules of high, medium, and low affinity. The approach offers a means for minimizing the volume of reagent required to functionalize the biosensor surface, while retaining compatibility with the microplate assay fluid-handling methods that are most commonly used in biological research.