Electrochemical microfluidic biosensor for the detection of nucleic acid sequences

A microfluidic biosensor with electrochemical detection for the quantification of nucleic acid sequences was developed. In contrast to most microbiosensors that are based on fluorescence for signal generation, it takes advantage of the simplicity and high sensitivity provided by an amperometric and coulorimetric detection system. An interdigitated ultramicroelectrode array (IDUA) was fabricated in a glass chip and integrated directly with microchannels made of poly(dimethylsiloxane) (PDMS). The assembly was packaged into a Plexiglas housing providing fluid and electrical connections. IDUAs were characterized amperometrically and using cyclic voltammetry with respect to static and dynamic responses for the presence of a reversible redox couple-potassium hexacyanoferrate (ii)/hexacyanoferrate (iii) (ferri/ferrocyanide). A combined concentration of 0.5 microM of ferro/ferricyanide was determined as lower limit of detection with a dynamic range of 5 orders of magnitude. Background signals were negligible and the IDUA responded in a highly reversible manner to the injection of various volumes and various concentrations of the electrochemical marker. For the detection of nucleic acid sequences, liposomes entrapping the electrochemical marker were tagged with a DNA probe, and superparamagnetic beads were coated with a second DNA probe. A single stranded DNA target sequence hybridized with both probes. The sandwich was captured in the microfluidic channel just upstream of the IDUA via a magnet located in the outside housing. Liposomes were lysed using a detergent and the amount of released ferro/ferricyanide was quantified while passing by the IDUA. Optimal location of the magnet with respect to the IDUA was investigated, the effect of dextran sulfate on the hybridization reaction was studied and the amount of magnetic beads used in the assay was optimized. A dose response curve using varying concentrations of target DNA molecules was carried out demonstrating a limit of detection at 1 fmol assay(-1) and a dynamic range between 1 and 50 fmol. The overall assay took 6 min to complete, plus 15-20 min of pre-incubation and required only a simple potentiostat for signal recording and interpretation.     

New detection modality for label-free quantification of DNA in biological samples via superparamagnetic bead aggregation

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, enabling label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without prior DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques.     

Cholera toxin subunit B detection in microfluidic devices

Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM₁ was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL⁻¹ for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.     

Magneto-mechanical detection of nucleic acids and telomerase activity in cancer cells

The ultra-sensitive magneto-mechanical detection of DNA, single-base-mismatches in nucleic acids, and the assay of telomerase activity are accomplished by monitoring the magnetically induced deflection of a cantilever functionalized with magnetic beads associated with the biosensing interface. The analyzed M13phi DNA hybridized with the nucleic acid-functionalized magnetic beads is replicated in the presence of dNTPs that include biotin-labeled dUTP. The resulting beads are attached to an avidin-coated cantilever, and the modified cantilever is deflected by an external magnetic field. Similarly, telomerization of nucleic acid-modified magnetic beads in the presence of dNTPs, biotin-labeled dUTP, and telomerase from cancer cell extracts and the subsequent association of the magnetic beads to the cantilever surface results in the lever deflection by an external magnetic field. M13phi DNA is sensed with a sensitivity limit of 7.1 x 10(-20) M by the magneto-mechanical detection method.     

Development of a lipase-based optical assay for detection of DNA

A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate a detectable readout the captured lipase was applied to an optical assay that takes advantage of the enzymatic activity of lipase. The assay applies p-nitrophenol octanoate (NPO) as the substrate and in the presence of lipase the ester is hydrolyzed to p-nitrophenolate which has a strong absorbance at 405 nm. The method provides detection a detection limit of 200 fmol target DNA and it was able to distinguish single base mismatches from the fully complementary target.     

Magnetic force-based multiplexed immunoassay using superparamagnetic nanoparticles in microfluidic channel

This paper describes a novel microfluidic immunoassay utilizing binding of superparamagnetic nanoparticles to beads and deflection of these beads in a magnetic field as the signal for measuring the presence of analyte. The superparamagnetic 50 nm nanoparticles and fluorescent 1 microm polystyrene beads are immobilized with specific antibodies. When target analytes react with the polystyrene beads and superparamagnetic nanoparticles simultaneously, the superparamagnetic nanoparticles can be attached onto the microbeads by the antigen-antibody complex. In the poly(dimethylsiloxane)(PDMS) microfluidic channel, only the microbeads conjugated with superparamagnetic nanoparticles by analytes consequently move to the high gradient magnetic fields under the specific applied magnetic field. In this study, the magnetic force-based microfluidic immunoassay is successfully applied to detect the rabbit IgG and mouse IgG as model analytes. The lowest concentration of rabbit IgG and mouse IgG measured over the background is 244 pg mL(-1) and 15.6 ng mL(-1), respectively. The velocities of microbeads conjugated with superparamagnetic nanoparticles are demonstrated by magnetic field gradients in microfluidic channels and compared with the calculated magnetic field gradients. Moreover, dual analyte detection in a single reaction is also performed by the fluorescent encoded microbeads in the microfluidic device. Detection range and lower detection limit can be controlled by the microbeads concentration and the higher magnetic field gradient.     

A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF^T1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF^T1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF^T1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.     

Superparamagnetic nanoparticle-polystyrene bead conjugates as pathogen capture mimics: a parametric study of factors affecting capture efficiency and specificity

There is currently significant interest in the miniaturization of disease detection platforms. As detection platforms decrease in size there is a need for the development of sample preparation protocols by which cells or biomarkers of interest can be concentrated from large volumes down to volumes more amenable to analysis within microfluidic devices. To address this issue, we present a series of magnetic confinement assays for polystyrene (PS) beads mediated through their covalent modification with a series of superparamagnetic nanoparticles, where the PS beads have many properties similar to bacteria, but are not pathogenic. The magnetic confinement of the PS beads is investigated as a function of (1) the overall nanoparticle size, (2) the loading of superparamagnetic content within the nanoparticle matrix, and (3) the viscosity and volume of the dispersion medium. We demonstrate that the time required for the magnetic capture of the PS beads by the superparamagnetic nanoparticles (1) decreases as the loading of superparamagnetic material into the nanoparticles increases and (2) increases as the viscosity and volume of the dispersion medium are increased. However, limitations in the magnetic confinement efficiency for the PS beads labeled with nanoparticles comprised of low loadings of superparamagnetic material can be overcome through the use of magnetic columns. These magnetic columns provide a practical and fast mode of sample preparation that should facilitate the magnetic concentration of cells and biomarkers from large volumes to volumes more amenable to incorporation into a microfluidic-based analysis system, where they can be analyzed/detected.     

An Optimized Bioassay for Mucin1 Detection in Serum Samples

Two simple and sensitive electrochemical approaches for Mucin1 (MUC1) tumor marker using magnetic beads coupling screen‐printed arrays were developed. The single‐use bioassays are based on a sandwich format in which aptamers or antibodies were coupled respectively to Streptavidin or Protein G‐modified magnetic beads. The bioreceptor‐modified beads are used to capture the MUC1 protein from the sample and sandwich assay is performed by the addition of a labeled secondary aptamer or antibody. The enzyme alkaline phosphatase and its substrate (1‐naphthyl phosphate) are then used for the electrochemical detection by differential pulse voltammetry (DPV). The analytical performances of the designed bioassays were compared in terms of sensitivity, selectivity and reproducibility. Using the optimized conditions, a linear range from 0 to 0.28 nM was obtained, with 0.19 nM LOD using antibody‐based and 0.07 nM LOD using aptamer‐based sandwich assay in MUC1 buffered solutions. The results also showed that the aptamer‐based approach exhibited higher selectivity for MUC1, allowing the detection of the protein in complex matrices. The developed aptasensor for MUC1 detection was applied on serum samples obtained from cancer patients, providing promising perspectives for clinical applications.     

Rapid superparamagnetic‐beads‐based automated immunoseparation of Zn‐proteins from Staphylococcus aureus with nanogram yield

Pathogenic bacteria have become a serious socio‐economic concern. Immunomagnetic separation‐based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles‐based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn‐proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn‐proteins by superparamagnetic beads modified with chicken anti‐Zn(II) antibody. S. aureus culture was treated with ZnCl₂. Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle–surface interactions were tested and associated protein profiles were identified using SDS‐PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn‐protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS‐PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.     

Electrochemical and Electrochemiluminescence Determination of Cancer Cells Based on Aptamers and Magnetic Beads

The electrochemical and electrochemiluminescence (ECL) detection of cell lines of Burkitt’s lymphoma (Ramos) by using magnetic beads as the separation tool and high‐affinity DNA aptamers for signal recognition is reported. Au nanoparticles (NPs) bifunctionalized with aptamers and CdS NPs were used for electrochemical signal amplification. The anodic stripping voltammetry technology employed for the analysis of cadmium ions dissolved from CdS NPs on the aggregates provided a means to quantify the amount of the target cells. This electrochemical method could respond down to 67 cancer cells per mL with a linear calibration range from 1.0×10² to 1.0×10⁵ cells mL^?1, which shows very high sensitivity. In addition, the assay was able to differentiate between target and control cells based on the aptamer used in the assay, indicating the wide applicability of the assay for diseased cell detection. ECL detection was also performed by functionalizing the signal DNA, which was complementary to the aptamer of the Ramos cells, with tris(2,2‐bipyridyl) ruthenium. The ECL intensity of the signal DNA, replaced by the target cells from the ECL probes, directly reflected the quantity of the amount of the cells. With the use of the developed ECL probe, a limit of detection as low as 89 Ramos cells per mL could be achieved. The proposed methods based on electrochemical and ECL should have wide applications in the diagnosis of cancers due to their high sensitivity, simplicity, and low cost.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

Magnetically trigged direct electrochemical detection of DNA hybridization using Au67 quantum dot as electrical tracer

A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.     

Prussian Blue Dispersed Sphere Catalytic Labels for Amplified Electronic Detection of DNA

A highly sensitive electrochemical DNA hybridization assay using Prussian blue (PB)-modified polymeric spheres as the oligonculeotide labeling tag is described. The sandwich assay relies on a secondary nucleic-acid probe functionalized with polystyrene beads loaded with numerous Prussian blue nanoparticles. The very strong catalytic activity of the captured PB 'artificial peroxidase' tag towards the reduction of hydrogen peroxide, along with the encapsulation of numerous catalytic particles onto polymeric beads, allows amperometric detection of the DNA target down to the 50 fM level (2.5 amol). Imaging and spectroscopic measurements are used to characterize the PB-tagged polystyrene beads. Such coupling of PB catalytic labels with polymeric carrier beads offers great promise for amplified transduction of different biomolecular interactions.     

Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

PCR-free MDR1 polymorphism identification by gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) represent the most abundant source of genetic variation in the human genome, and they can be linked to genetic susceptibilities or varied pharmaceutical responses. Established SNP detection techniques are mainly PCR-based, which means that they involve complex, labor-intensive procedures, are easy contaminated, and can give false-positive results. Therefore, we have developed a simple and rapid MS-based disulfide barcode methodology that relies on magnifying the signal from a dual-modified gold nanoparticle. This approach permits direct SNP genotyping of total human genomic DNA without the need for primer-mediated enzymatic amplification. Disulfides that are attached to the gold nanoparticle serve as a “barcode” that allows different sequences to be discerned using MS detection. Specificity is based on two sequential oligonucleotide hybridizations, which include two steps: the first is the capture of the target by gene-specific probes immobilized onto magnetic beads; the second is the recognition of gold nanoparticles functionalized with allele-specific oligonucleotides. The sensitivity of this new method reaches down to the 0.1 fM range, thus approaching that of PCR. The feasability of this SNP identification methodology based on an MS-based disulfide barcode assay was demonstrated by applying it to genomic DNA samples representing all possible genotypes of the SNPs G2677T and C3435T in the human MDR1 gene. Due to its great advantage—the ability to perform SNP typing without the use of PCR—the assay was found to be simple, rapid and robust, and so may be highly suited to routine clinical detection as well as basic medical research.     

Aptamer‐Based SERS Assay of ATP and Lysozyme by Using Primer Self‐Generation

A simple bifunctional surface‐enhanced Raman scattering (SERS) assay based on primer self‐generation strand‐displacement polymerization (PS‐SDP) is developed to detect small molecules or proteins in parallel. Triphosphate (ATP) and lysozyme are used as the models of small molecules and proteins. Compared to traditional bifunctional methods, the method possesses some remarkable features as follows: 1) by virtue of the simple PS‐SDP reaction, a bifunctional aptamer assembly binding of trigger 1 and trigger 2 was used as a functional structure for the simultaneous sensing of ATP or lysozyme. 2) The concept of isothermal amplification bifunctional detection has been first introduced into SERS biosensing applications as a signal‐amplification tool. 3) The problem of high background induced by excess bio‐barcodes is circumvented by using magnetic beads (MBs) as the carrier of signal‐output products and massive of hairpin DNA binding with SERS active bio‐barcodes relied on Au nanoparticles (Au NPs), SERS signal is significantly enhanced. Overall, with multiple amplification steps and one magnetic‐separation procedure, this flexible biosensing system exhibited not only high sensitivity and specificity, with the detection limits of ATP and lysozyme of 0.05 nM and 10 fM , respectively.     

Aptamer‐based thrombin assay on microfluidic platform

A facile and sensitive aptamer‐based protocol has been developed for protein assay on microfluidic platform with fluorescence detection using an off‐chip microarray scanner. Aptamer‐functionalized magnetic beads were used to capture thrombin that binds to a second aptamer fluorescently labeled by Cy3. Experimental conditions, such as incubation time and temperature, washing time, interfering proteins, and aptamer, etc., were optimized for the microchip method. This work demonstrated there was a good relationship between fluorescence intensity and thrombin concentration in the range of 65–1000 ng/mL with the RSD less than 8%. Notably, an analysis only needs 1 μL volume of sample injection and this system can capture extremely tiny amount thrombin (0.4 fmol). This method has been successfully applied to assay of thrombin in human serum with the recovery of 79.74–95.94%.     

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.