Microfluidic system for DNA sequence detection

Most challenging in the development of DNA sensors is the ability to distinguish between fully complementary target ssDNA (single-strand DNA) and 1-mismatch ssDNA. To deal with this problem, we performed impedance spectroscopy on DNA-functionalized nanocrystalline diamond (NCD) layers during hybridization and denaturation. In both reactions, a difference in behavior was observed for 1-mismatch target DNA and complementary target DNA in real-time. During real-time hybridization, a decrease of the impedance was observed at lower frequencies when the complementary target DNA was added, while the addition of 1-mismatch target ssDNA caused no significant change. Fitting these results to an electrical circuit demonstrates that this is correlated with a decrease of the depletion zone in the space charge region of the diamond. During real-time denaturation, differentiation between 1-mismatch and complementary target DNA was possible at higher frequencies. Denaturation of complementary DNA showed the longest exponential decay time of the impedance, while the decay time during 1-mismatch denaturation was the shortest. The real-time hybridization and denaturation experiments were carried out on different NCD samples in various buffer solutions at temperatures between 20 and 80 degrees C. It was revealed that the best results were obtained using a Microhyb hybridization buffer at 80 degrees C and 10x PCR buffer at 30 degrees C for hybridization and 0.1 M NaOH at temperatures above 40 degrees C for denaturation. We demonstrate that the combination of real-time hybridization spectra and real-time denaturation spectra yield important information on the type of target. This approach may allow a reliable identification of the mismatch sequence, which is the most biologically relevant.

A morphologically complex cellulosic substance (e.g., commercial filter paper) was employed as a substrate for DNA immobilization and successive recognition. A uniform ultrathin zirconia gel film was first deposited on each cellulose nanofiber in bulk filter paper by a facile sol-gel process. Relying on the large surface area of filter paper and the strong affinity of zirconia for the phosphate group, terminal-phosphate probe DNA was abundantly immobilized on the zirconia-modified filter paper so as to convert the composite to a biofunctional material for the sensitive and repetitive recognition of the corresponding complementary target DNA on the nanomolar level. By contrast, in spite of the viability of the immobilization of the probe DNA and the recognition of target DNA on the quartz plate, the amount of captured probe DNA or recognized target DNA on such a flat substrate was much less than that captured or recognized on filter paper, resulting in a relatively insensitive recognition event. Moreover, control experiments on bare filter paper (without a zirconia nanocoating) suggested that the zirconia gel film was essential to probe DNA immobilization and subsequent target DNA recognition.

Detection of disease-related gene expression by DNA hybridization is a useful diagnostic method. In this study a monolayer graphene field effect transistor (GFET) was fabricated for the detection of a particular single-stranded DNA (target DNA). The probe DNA, which is a single-stranded DNA with a complementary nucleotide sequence, was directly immobilized onto the graphene surface without any linker. The VDirac was shifted to the negative direction in the probe DNA immobilization. A further shift of VDirac in the negative direction was observed when the target DNA was applied to GFET, but no shift was observed upon the application of non-complementary mismatched DNA. Direct immobilization of double-stranded DNA onto the graphene surface also shifted the VDirac in the negative direction to the same extent as that of the shift induced by the immobilization of probe DNA and following target DNA application. These results suggest that the further shift of VDirac after application of the target DNA to the GFET was caused by the hybridization between the probe DNA and target DNA.

Programmable graphene-based microfluidic sensor for DNA detection

The dominant physical transport processes are analyzed in a free-surface microfluidic and surface-enhanced Raman spectroscopy (SERS) chemical detection system. The analysis describes the characteristic fluid dynamics and mass transport effects occurring in a microfluidic detection system whose analyte absorption and concentration capability is designed to operate on principles inspired by canine olfaction. The detection system provides continuous, real-time monitoring of particular vapor-phase analytes at concentrations of 1 ppb. The system is designed with a large free-surface-to-volume ratio microfluidic channel which allows for polar or hydrophilic airborne analytes to readily be partitioned from the surrounding gas phase into the aqueous phase for detection. The microfluidic stream can concentrate certain molecules by up to 6 orders of magnitude, and SERS can enhance the Raman signal by 9-10 orders of magnitude for molecules residing in the so-called SERS "hot spots", providing extremely high detection sensitivity. The resulting vibrational spectra are sufficiently specific to identify the detected analyte unambiguously. Detection performance was demonstrated using a nominal 1 ppb, 2,4-dinitrotoluene (2,4-DNT) vapor stream entrained within N(2) gas. Applications to homeland security arise from the system's high sensitivity and its ability to provide highly reproducible, continuous chemical detection monitoring with minimal sampling requirements.

This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.

Early detection of pathogens in high-value agricultural species is crucial. Therefore, many methods which can detect agricultural pathogens to prevent economic loss have been developed. Among them, immunoassays, nucleic acid hybridization and polymerase chain reaction have been demonstrated to detect pathogens in Phalaenopsis orchid successfully with satisfactory sensitivity and specificity. However, above-mentioned methods all have some disadvantages including lengthy process or requiring specialized laboratory facilities and well-trained technicians. The current study therefore presents an integrated micro fluidic system for rapid and automatic detection of pathogens in agricultural species. The entire procedure, including pathogen-specific ribonucleic acid (RNA) purification, nucleic acid amplification using reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection, can be automatically performed on a single chip within 65 minutes. Moreover, the detection module can be interchanged between a fluorescent detector and a turbidity detector, depending on the need of the operator. Furthermore, the developed system can detect pathogens directly from fresh agriculture tissues such as leaves and flowers of the Phalaneopsis orchids. This is the first time that an interchangeable integrated micro fluidic system for the detection of Phalaneopsis orchids has been demonstrated. Some of the most prevalent Phalaenopsis orchid pathogens., such as Cymbidium mosaic virus (CymMV) and Tomato spotted wilt virus (TSWV) for Phalaneopsis orchids were used in the current study to demonstrate the capabilities of the developed system. It is concluded that this system with dual detection units can directly detect pathogens from crude agricultural materials successfully.

This research aims to develop DNA chip array without an indicator. We fabricated microelectrode array by photolithography technology. Several DNA probes were immobilized on an electrode. Then, indicator-free target DNA was hybridized by an electrical force and measured electrochemically. Cyclic-voltammograms (CVs) showed a difference between DNA probe and mismatched DNA in an anodic peak. Immobilization of probe DNA and hybridization of target DNA could be confirmed by fluorescent. This indicator-free DNA chip microarray resulted in the sequence-specific detection of the target DNA quantitatively ranging from <TEX>$10^{-18}\;M\;to\;10^{-5}$</TEX> M in the buffer solution. This indicator-free DNA chip resulted in a sequence-specific detection of the target DNA.

Over the past few years the concept of using magnetic field sensors for biological applications in particular, the development of magnetoresistive biochips and biosensors, has generated increasing interest from laboratories and companies. A spin-valve sensor based biochip was used to detect cystic fibrosis related DNA targets for the purpose of developing an affordable diagnostic chip and detection system. The strategy is based on the AC magnetic field focusing technique. This method consists of the attraction, concentration and manipulation of magnetically-labelled target DNA within on-chip u-shaped current line regions surface functionalized with a cystic fibrosis-related DNA probe. Cystic fibrosis related probes were immobilized on the oxide surface and 250 nm diameter non-remanent magnetic particles were functionalized with cystic fibrosis related DNA targets complementary or non-complementary to the immobilized probes. The hybridization of the target is detected using a u-shaped spin-valve sensor fabricated within the line structure. The proximity of probe and target at the spin-valve sensor surface promotes the hybridization of complementary DNA strands. In this way, hybridization occurs in relatively short times, (5-25 minutes), in comparison with conventional hybridization approaches (3 to 12 hours), as limited by diffusion of the target DNA in solution. Magnetic labels bound to the sensor surface through the hybridization of complementary DNA strands have a magnetic stray field that changes the resistance of sensors enabling detection of the hybridization in real-time. Results show a discernable difference in sensor response after washing when using complementary or non-complementary DNA targets. The use of complementary target DNA resulted in distinct hybridization signals and the binding of the particles in the sensor area was verified by visual inspection. In addition, it was observed that hybridization signals decreased slightly after the more stringent wash indicating that non-specifically or weakly bound labels were washed away. The use of non-complementary target DNA resulted in negligible sensor response after washing and no particles were observed in the sensor area.

An electrochemical DNA sensor for Legionella pneumophila detection was constructed using O2 plasma functionalized multi-walled carbon nanotube (MWCNT) film as a working electrode (WE). The cyclic voltammetry (CV) results revealed that the electrocatalytic activity of plasma functionalized MWCNT (pf-MWCNT) significantly changed depending on O2 plasma treatment time due to some oxygen containing functional groups on the pf-MWCNT surface. Scanning electron microscope (SEM) images and X-ray photoelectron spectroscopy (XPS) spectra were also presented the changes of their surface morphologies and oxygen composition before and after plasma treatment. From a comparison study, it was found that the pf-MWCNT WEs had higher electrocatalytic activity and more capability of probe DNA immobilization: therefore, electrochemical signal changes by probe DNA immobilization and hybridization on pf-MWCNT WEs were larger than on Au WEs. The pf-MWCNT based DNA sensor was able to detect a concentration range of 10 pM–100 nM of target DNA to detect L. pneumophila.

A label-free two-dimensional colorimetric DNA sensor is reported. This sensor is based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes induced by the molecular hybridization of single-stranded oligonucleotide probes and their complementary single-stranded DNA targets. To detect the aggregation, we have developed a new detection method based on the selective desorption of nonaggregated nanoparticles. We will show here that this detection method is highly specific and allows the quantification of the DNA targets.

Gene detection has important applications in biology, biomedical engineering, clinical, environmental, and marine fields. Rapid, sensitive, and selective recognition of specific genes is essential in practical applications. In this study, we describe a facile and sensitive DNA sensing platform for specific and quantitative detection of Heterosigma akashiwo, which is one of the causative agents of red tides. Fast and sensitive detection is achieved by using chemically synthesized graphene oxide (GO) nanosheets. Probe DNA is designed according to the specific DNA fragments of harmful algae and labeled with fluorescent molecules FAM (fluorescein-based dye). GO nanosheet solution is made, in which the strong interaction between FAM-labeled probe and GO nanosheets keeps them in close proximity, facilitating the fluorescence quenching of the fluorophore by GO nanosheets. In the presence of a complementary target DNA, the FAM-labeled DNA probe and the target DNA hybridize and desorb from the surface of GO nanosheets, resulting in restoration of fluorophore fluorescence. The concentration of target DNA fragments is analyzed by the fluorescence intensity at ~ 520nm with emission wavelength of 480nm. The sensitive detecting platform achieved stable measurement of 1pM specific genes from Heterosigma akashiwo. Our GO nanosheet-based DNA-sensing platform performs fast and sensitive detection of trace amount of DNA, and enables quantitative recognition of harmful algae, which has promising applications in real-time monitoring in the marine environment of red tide generative dynamics, allowing effective control, particularly in relation to marine aquaculture.

In this paper, recent research activities at the Microsystems and BioMEMS Laboratory at the University of Cincinnati (UC) will be presented, which includes handheld biochemical detection systems with sampling and analysis capabilities and plastic based disposable biochips for clinical diagnostics. In the past few years, numerous microfluidic prototype devices and systems have been developed, specifically for biochemical warfare detection systems and portable diagnostic applications. BioMEMS-research team at the University of Cincinnati has been working on the development of a remotely accessible generic microfluidic system for biochemical detection and biomedical analysis, based on the concepts of both surface-mountable microfluidic motherboards and electrochemical detection techniques. The goal of this work is to develop a generic MEMS-based microfluidic system and to apply the fluidic system to detect bio-molecules such as specific proteins and/or antigens in liquid samples.

A simple and sensitive method for electrochemical detection of DNA was designed. This DNA sensor was based on a "sandwich" detection strategy, which involved a long capture probe DNA immobilized on glassy carbon electrodes that flanked both the reference DNA and target DNA. Electrochemical signals were measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using aquadichloro(benzimidazole)-copper(II), Cu(bzim)(H(2)O)Cl(2), as an electroactive indicator. An improving amount of Cu(bzim)(H(2)O)Cl(2) was interacted with the hybrid DNA via the incorporation of a long-probe DNA and a reference DNA in this sensor. As a result of this effect, this sensor design significantly enhanced the sensitivity. With 48-mer probe DNA and 27-mer reference DNA, the proposed method could be used for detection of 21-mer ssDNA ranging from 1.32 x 10(-7) to 2.52 x 10(-6) M with a detection limit of 2.94 x 10(-8) M. Electrochemical DNA biosensors were also developed using the same long-probe sequence as the target sequence with the novel hybridization indicator, Cu(bzim) (H(2)O)Cl(2). The detection limits for the complementary 21-mer target and 27-mer target were 9.52 x 10(-8) M and 5.81 x 10(-8) M, respectively. The results showed that the sensor with long-probe DNA and reference DNA is far more sensitive than that with nonswitch assay.

Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe