On-chip Hybridization Research Articles

Development of an Integrated CMUTs-Based Resonant Biosensor for Label-Free Detection of DNA with Improved Selectivity by Ethylene-Glycol Alkanethiols

Gravimetric resonant-inspired biosensors have attracted increasing attention in industrial and point-of-care applications, enabling label-free detection of biomarkers such as DNA and antibodies. Capacitive micromachined ultrasonic transducers (CMUTs) are promising tools for developing miniaturized high-performance biosensing complementary metal–oxide–silicon (CMOS) platforms. However, their operability is limited by inefficient functionalization, aggregation, crosstalk in the buffer, and the requirement for an external high-voltage (HV) power supply. In this study, we aimed to propose a CMUTs-based resonant biosensor integrated with a CMOS front–end interface coupled with ethylene–glycol alkanethiols to detect single-stranded DNA oligonucleotides with large specificity. The topography of the functionalized surface was characterized by energy-dispersive X-ray microanalysis. Improved selectivity for on-chip hybridization was demonstrated by comparing complementary and non-complementary single-stranded DNA oligonucleotides using fluorescence imaging technology. The sensor array was further characterized using a five-element lumped equivalent model. The 4 mm2 application-specific integrated circuit chip was designed and developed through 0.18 μm HV bipolar-CMOS-double diffused metal–oxide–silicon (DMOS) technology (BCD) to generate on-chip 20 V HV boosting and to track feedback frequency under a standard 1.8 V supply, with a total power consumption of 3.8 mW in a continuous mode. The measured results indicated a detection sensitivity of 7.943 × 10−3 μmol∙L−1∙Hz−1 over a concentration range of 1 to 100 μmol∙L−1. In conclusion, the label-free biosensing of DNA under dry conditions was successfully demonstrated using a microfabricated CMUT array with a 2 MHz frequency on CMOS electronics with an internal HV supplier. Moreover, ethylene–glycol alkanethiols successfully deposited self-assembled monolayers on aluminum electrodes, which has never been attempted thus far on CMUTs, to enhance the selectivity of bio-functionalization. The findings of this study indicate the possibility of full-on-chip DNA biosensing with CMUTs.

P.325 Reward anticipation processing in major depressive disorder and prediction of treatment response - a functional magnetic resonance imaging study

DNA hybridization kinetics has been playing a critical role in molecular diagnostics for binding discrimination, but its study on digital microfluidic (DMF) systems is ultimately restrained by the laminar flow condition. The kinetic mixing technique is widely employed to ensure a fast reaction rate, but poses intrinsic risk in cross contamination and exhibits instable fluorescence intensity during the droplet transportation. While the electrothermal technique can provide stationary droplet mixing through the established thermal gradient within the hybridization solution, the significant increase in the droplet temperature will inevitably undermine the hybridization equilibrium and jeopardize the binding discrimination. To enhance the hybridization efficiency while ensuring a stable droplet temperature (within ±0.1℃), this paper presents a DMF platform that can perform isothermal hydrodynamic-flow-enhanced droplet mixing. Specifically, with a single electrode, droplet-boundary oscillation under a slow AC actuation is studied for improving the reaction rate. The dependencies between the mixing efficiency and the actuation voltage, actuation frequency and the spacer thickness are also systematically studied. Reliable mixing efficiency improvement is further validated over a wide range of solute concentrations. The results from real-time on-chip DNA hybridization kinetics with stationary droplets using the complete sandwiched DMF system shows that the proposed rapid mixer can achieve the same hybridization equilibrium with >13 times faster reaction rate when compared to the reference one through pure diffusion, while preventing biased hybridization kinetics as demonstrated in the electrothermal technique.

Hydrodynamic-flow-enhanced rapid mixer for isothermal DNA hybridization kinetics analysis on digital microfluidics platform

DNA hybridization kinetics has been playing a critical role in molecular diagnostics for binding discrimination, but its study on digital microfluidic (DMF) systems is ultimately restrained by the laminar flow condition. The kinetic mixing technique is widely employed to ensure a fast reaction rate, but poses intrinsic risk in cross contamination and exhibits instable fluorescence intensity during the droplet transportation. While the electrothermal technique can provide stationary droplet mixing through the established thermal gradient within the hybridization solution, the significant increase in the droplet temperature will inevitably undermine the hybridization equilibrium and jeopardize the binding discrimination. To enhance the hybridization efficiency while ensuring a stable droplet temperature (within ±0.1℃), this paper presents a DMF platform that can perform isothermal hydrodynamic-flow-enhanced droplet mixing. Specifically, with a single electrode, droplet-boundary oscillation under a slow AC actuation is studied for improving the reaction rate. The dependencies between the mixing efficiency and the actuation voltage, actuation frequency and the spacer thickness are also systematically studied. Reliable mixing efficiency improvement is further validated over a wide range of solute concentrations. The results from real-time on-chip DNA hybridization kinetics with stationary droplets using the complete sandwiched DMF system shows that the proposed rapid mixer can achieve the same hybridization equilibrium with >13 times faster reaction rate when compared to the reference one through pure diffusion, while preventing biased hybridization kinetics as demonstrated in the electrothermal technique.

Surface regeneration and reusability of label-free DNA biosensors based on weak polyelectrolyte-modified capacitive field-effect structures

The reusability of capacitive field-effect electrolyte-insulator-semiconductor (EIS) sensors modified with a cationic weak polyelectrolyte (poly(allylamine hydrochloride) (PAH)) for the label-free electrical detection of single-stranded DNA (ssDNA), in-solution- and on-chip-hybridized double-stranded DNA (dsDNA) has been studied. It has been demonstrated that via simply regeneration of the gate surface of the EIS sensor by means of an electrostatic adsorption of a new PAH layer, the same biosensor can be reused for at least five DNA-detection measurements. Because of the reversal of the charge sign of the outermost layer after each surface modification with the cationic PAH or negatively charged DNA molecules, the EIS-biosensor signal exhibits a zigzag-like behavior. The amplitude of the signal changes has a tendency to decrease with increasing number of macromolecular layers. The direction of the EIS-signal shifts can serve as an indicator for a successful DNA-immobilization or -hybridization process. In addition, we observed that the EIS-signal changes induced by each surface-modification step (PAH adsorption, immobilization of ssDNA or dsDNA molecules and on-chip hybridization of complementary target cDNA) is decreased with increasing the ionic strength of the measurement solution, due to the more efficient macromolecular charge-screening by counter ions. The results of field-effect experiments were supported by fluorescence-intensity measurements of the PAH- or DNA-modified EIS surface using various fluorescence dyes.

Optimizing the Toehold Strategy of On-Chip Nucleic Acid Hybridization Probe for the Discrimination of Single Nucleotide Polymorphism.

The synthetic DNA hybridization probe has proved its importance in biology and biotechnology. In this study, taking advantage of a novel analytical technique called dual polarization interferometry (DPI), the influence of the toehold strategy of on-chip DNA hybridization probe on the discrimination of single nucleotide polymorphism (SNP) was investigated. Through adjusting the toehold length, the toehold strategies of on-chip toehold exchange probe were thoroughly optimized. For the "6/5" probe, an optimal discrimination factor of 78% against the spurious target was achieved. Moreover, the ability of the on-chip probe in SNP discrimination was significantly enhanced compared to its pure solution counterpart. This simple and rapid detection method for SNP discrimination based on the on-chip toehold exchange probe will show great potential in disease diagnosis.

Potentialities of MicroRNA Diagnosis in Patients with Bladder Cancer.

Despite promising vista of the use of microRNA in molecular diagnosis of bladder cancer, there are few data on their expression profiles, which impedes assessment of diagnostic value of these marker molecules. In this study, suppression subtractive hybridization, on-chip hybridization, and high-throughput deep sequencing focused on profiling microRNA and assessing the diagnostic value of revealed marker molecules.

Graphene quantum dot-based on-chip electrochemical DNA hybridization sensor for pancreatic cancer

Graphene quantum dot-based on-chip electrochemical DNA hybridization sensor for pancreatic cancer

A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules

Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39fM (831 molecules in 35.4nl assay volume) for target DNA and 16fM (338 molecules) for target RNA after 1h on-chip hybridization.

Eggshell membrane: A natural substrate for immobilization and detection of DNA

Chemically modified eggshell membranes (ESM) have been explored as potentially novel platforms for immobilization of oligonucleotides and subsequent detection of target DNA. The fibrous network of the native ESM as well those functionalized with acetic acid or n-butyl acetate has been examined by field-emission scanning electron microscopy (FESEM). The formation of surface functional moieties has been confirmed by Fourier-transform infrared spectroscopy (FTIR). DNA molecules, with an end terminal −NH2 group (at 5′ end) have been immobilized on the chemically modified ESM surface. The effect of surface modification on the DNA immobilization efficiency has been investigated using fluorescence microscopy and atomic force microscopy (AFM). The above studies concurrently suggest that functionalization of ESM with n-butyl acetate causes a better homogeneity of the DNA probes on the membrane surface. On-chip hybridization of the target DNA with the surface bound capture probes has been performed on the functionalized membranes. It is observed that n-butyl acetate modification of ESM pushes the limit of detection (LOD) of the DNA sensors by at least an order of magnitude compared to the other modification method.

KRAS mutation screening by chip-based DNA hybridization--a further step towards personalized oncology.

The use of predictive biomarkers can help to improve therapeutic options for the individual cancer patient. For the treatment of colon cancer patients with anti-EGFR-based drugs, the KRAS mutation status has to be determined to pre-select responders that will benefit from this medication. Amongst others, array-based tests have been established for profiling of the KRAS mutation status. Within this article we describe an on-chip hybridization technique to screen therapeutic relevant KRAS codon 12 mutations. The DNA chip-based platform enables the reliable discrimination of selected mutations by allele-specific hybridization. Here, silver deposits represent robust endpoint signals that allow for a simple naked eye rating. With the here presented assay concept a precise identification of heterozygous and homozygous KRAS mutations, even against a background of up to 95% wild-type DNA, was realizable. The applicability of the test was successfully proven for various cancer cell lines as well as clinical tumour samples. Thus, the chip-based DNA hybridization technique seems to be a promising tool for KRAS mutation analysis to further improve personalized cancer treatment.

On-site detection of Phytophthora spp.—single-stranded target DNA as the limiting factor to improve on-chip hybridization

We report on a lab-on-a-chip approach for on-site detection of Phytophthora species that allows visual signal readout. The results demonstrate the significance of single-stranded DNA (ssDNA) generation in terms of improving the intensity of the hybridization signal and to improve the reliability of the method. Conventional PCR with subsequent heat denaturation, sodium hydroxide-based denaturation, lambda exonuclease digestion and two asymmetric PCR methods were investigated for the species P. fragariae, P. kernoviae, and P. ramorum. The positioning of the capture probe within the amplified yeast GTP-binding protein (YPT1) target DNA was also of interest because it significantly influences the intensity of the signal. Statistical tests were used to validate the impact of the ssDNA generation methods and the capture-target probe position. The single-stranded target DNA generated by Linear-After-The-Exponential PCR (LATE-PCR) was found to produce signal intensities comparable to post-PCR exonuclease treatment. The LATE-PCR is the best method for the on-site detection of Phytophthora because the enzymatic digestion after PCR is more laborious and time-consuming.

Integrated Printed Circuit Board Device for Cell Lysis and Nucleic Acid Extraction

Preparation of raw, untreated biological samples remains a major challenge in microfluidics. We present a novel microfluidic device based on the integration of printed circuit boards and an isotachophoresis assay for sample preparation of nucleic acids from biological samples. The device has integrated resistive heaters and temperature sensors as well as a 70 μm × 300 μm × 3.7 cm microfluidic channel connecting two 15 μL reservoirs. We demonstrated this device by extracting pathogenic nucleic acids from 1 μL dispensed volume of whole blood spiked with Plasmodium falciparum. We dispensed whole blood directly onto an on-chip reservoir, and the system's integrated heaters simultaneously lysed and mixed the sample. We used isotachophoresis to extract the nucleic acids into a secondary buffer via isotachophoresis. We analyzed the convective mixing action with micro particle image velocimetry (micro-PIV) and verified the purity and amount of extracted nucleic acids using off-chip quantitative polymerase chain reaction (PCR). We achieved a clinically relevant limit of detection of 500 parasites per microliter. The system has no moving parts, and the process is potentially compatible with a wide range of on-chip hybridization or amplification assays.

Rapid screening of phenylketonuria using a CD microfluidic device

We herein present a compact disc (CD) microfluidic chip based hybridization assay for phenylketonuria (PKU) screening. This CD chip is composed of a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a glass bottom layer with hydrogel pad conjugated DNA oligonucleotides. Reciprocating flow was generated on the CD chip through a simple rotation-pause operation to facilitate rapid DNA hybridization. When rotated the CD chip, the sample solution was driven into the hybridization channel by centrifugal force. When stopped the CD chip, the sample plug was pulled backward through the channel by capillary force. The hybridization assay was firstly validated with control samples and was then used to analyze 30 clinical samples from pregnant women with suspected PKU fetus. The on-chip DNA hybridization was completed in 15 min with a sample consumption as low as 1.5 μL, and the limit-of-detection (LOD) of DNA template was 0.7 ng/μL. Among the 30 samples tested, V245V mutation was identified in 4 cases while R243Q mutation was detected in one case. Results of the hybridization assay were confirmed by DNA sequencing. This CD-chip based hybridization assay features short analysis time, simple operation and low cost, thus has the potential to serve as the tool for PKU screening.

Development of a compact disk type microfluidic chip based on DNA hybridization for phenylketonuria screening

Objective To develop a phenylketonuria (PKU) screening method based on a compact disk (CD) type microfluidic chip capable of generating reciprocating flow within the microchannels that facilitate rapid DNA hybridization. Methods This microfluidic device consists of a two-layer structure: a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a bottom glass layer with immobilized hydrogel conjugated DNA arrays. The DNA arrays included R243Q, V245V and the blank control probes. When the CD device was spun, the PCR products were driven into the hybridization channel by centrifugal force. When the rotation of the CD device was stopped, capillary force pulled the PCR products solution to flow back to the channel. After the on-chip hybridization, the hybridization signals were captured on a fluorescence microscope. The specificity, detection limitation and reproducibility of this device were evaluated. Thirty DNA samples from pregnant women with suspected PKU were detected by this device.Then the results were compared with DNA sequencing results. Results With the compact disk type microfluidic chip, the hybridization time could be reduced to 15 min, sample consume could be as low as 1. 5 μl and the detection limitation was 0. 7 ng/μl. With the chip based method, samples of PKU patients and healthy controls were detected and the results were consistent with DNA sequencing results. Five different batches of chips and five micro-channels of each chip were selected to test one PKU patients with V245V mutation. All the results were positive, indicating good reproducibility. Four cases of V245V mutation and 1 case of R243Q mutation were found in 30 suspected PKU carried pregnant women. Conclusion The compact disk microfluidic device has advantages of simple, rapid and highly sensitive, thus is well suited to PKU screening. Key words: Phenylketonuria; Phenylalanine hydroxylase; Microfluidic analytical techniques; Nucleic acid hybridization

Label-free low-cost disposable DNA hybridization detection systems using organic TFTs

Organic thin film transistors (OTFT) are potentially very attractive for making portable and disposable DNA hybridization detection toolkits because of their ultra-low-cost manufacturing and potentially high sensitivity. To make a self-supported DNA sensor, we herein report an OTFT-based label-free DNA hybridization detection system integrating electrically read DNA hybridization sensors and microfluidic delivery channels. We previously reported DNA-doped OTFTs acting as electrically read DNA sensors. In this article, we first study physics of DNA sensitivity of OTFTs, clarifying the doping mechanism. The article also presents a method of verification of the immobilization and presence of DNA on the surface using TOF-SIMS analysis. As a further step, sources of variation of measured data and methods of minimization are discussed using a novel photolithography-based microfluidics fabrication method, directly enabling on-chip hybridization and self-supported DNA detection. By integrating the sensors with microfluidics, for the first time, we demonstrate necessary technologies required to realize disposable, rapid turn-around tools for field-deployable genetic diagnosis.

Femtomolar limit of detection with a magnetoresistive biochip

In this paper the biological limit of detection of a spin-valve-based magnetoresistive biochip applied to the detection of 20mer ssDNA hybridization events is presented. Two reactional variables and their impact on the biomolecular recognition efficiency are discussed. Both the influence of a 250nm diameter magnetic particle attached to the target molecule during the hybridization event and the effect of a magnetic focusing system in the hybridization of pre-labeled target DNA (assisted hybridization) are addressed. The particles carrying the target molecules are attracted to the probe active sensor sites by applying a 40mA DC current on U-shaped aluminium current lines. Experiments comparing pre-hybridization versus post-hybridization magnetic labeling and passive versus magnetically assisted hybridization were conducted. The efficiency of a passive hybridization is reduced by about 50% when constrained to the operational conditions (sample volume, reaction time, temperature and magnetic label) of an on-chip real-time hybridization assay. This reduction has shown to be constant and independent from the initial target concentration. Conversely, the presence of the magnetic label improved the limit of detection when a magnetically assisted hybridization was performed. The use of a labeled target focusing system has permitted a gain of three orders of magnitude (from 1pM down to 1fM) in the sensitivity of the device, as compared with passive, diffusion-controlled hybridization.

Oligonucleotide hybridization and free-solution electrokinetic separation in a nanofluidic device

There is significant interest in developing on-chip DNA hybridization assays to leverage the advantages of lab-on-a-chip systems, which include smaller sample and reagent volumes, faster processing speeds, and greater opportunities for large-scale integration. While much research has explored ways to integrate DNA microarrays on-chip, little work has been done to incorporate hybridization with existing microscale separation platforms. We present the first separation of single-stranded and double-stranded oligonucleotides in a nanofluidic device. We couple this separation with free-solution hybridization to develop a simple, electrokinetic technique that detects DNA hybridization without sample labeling. The technique is used both to detect target DNA sequences and to quantitatively measure hybridization kinetics. To demonstrate the method, we measured the second order reaction coefficient of complementary 20-mer oligonucleotides as a function of sodium ion concentration, which ranged from 0.0048 mol(-1).sec(-1) at 5 mM sodium to 0.42 mol(-1).sec(-1) at 50 mM. We also distinguished between a pair of complementary oligonucleotides and a pair with a single nucleotide mismatch, observing a two-fold difference in hybridization rate. Additionally, we observed a relative change in the mobility of single-stranded and double-stranded DNA with increasing sodium concentration, suggesting that our device may provide a useful platform for studying biomolecule transport in nanochannels.

SPR study of DNA hybridization with DNA and PNA probes under stringent conditions

Surface plasmon resonance (SPR) spectroscopy has been used for studying on-chip DNA hybridization to a PNA probe and its counterpart DNA probe of a 22-mer sequence. Two stringency control strategies are used for single base mismatch discrimination, namely (1) adding a denaturant, i.e. formamide (FA), into hybridization buffer and (2) coupling negative potentials for selective dehybridization of mismatch DNA. These two strategies have either not been used before or been less-well studied in SPR detection. An end-point SPR measurement protocol (no real-time hybridization profile recorded) is developed for detecting DNA hybridization in the presence of FA, to circumvent the problem that the refractive index of FA is out of the detectable range of the SPR equipment. The missing of real-time measurement of hybridization profile is compensated with QCM measurement. Under optimal conditions, i.e. 10 mM PBS with 30% FA and 1 mM PBS with 50% FA, single base mismatch DNA is detected with 1.7 and 2.8 times less hybridization signals compared with the perfect match DNA, with the DNA probe and PNA probe, respectively. Under negative potential of −0.2 to −0.4 V (vs. Ag/AgCl), mismatch DNA dissociates more than perfect match DNA by 1.7–2.5 times from the DNA probe and 2.1–3.5 times from the PNA probe. The higher mismatch discrimination efficiency of the PNA probe under stringent conditions would be attributable to its higher intrinsic sequence selectivity.

Metal Cation-Induced Deformation of DNA Self-Assembled Monolayers on Silicon: Vibrational Sum Frequency Generation Spectroscopy

Nucleic acids possess charged phosphate groups in their backbones, which require counterions to reduce the repulsive Coulombic interactions between the strands. Herein we report how different mono- and divalent metal cations influence the molecular orientations of DNA molecules on silicon surfaces upon immobilization and hybridization. Our sum frequency generation (SFG) spectroscopy studies demonstrated that the degree of conformational variation of DNA self-assembled monolayers on silicon depends on the type of metal cations present. The molecular orientation change of immobilized single-stranded oligonucleotides correlates with DNA-cation affinity (Mg(2+) > Ca(2+) > K(+) approximately Na(+)): metal cations with the strongest affinity disrupt the structure of the underlying linker monolayer the most. Upon hybridization the trend is reversed, which is attributed to the greater ability of divalent cations to mask the negative charges on the DNA backbone. These findings provide useful information for the construction of more sensitive DNA biosensors, particularly the optimization of on-chip hybridization performance.

16S rRNA Sequence-based Rapid and Sensitive Detection of Aquatic Bacteria by On-chip Hybridization Following Multiplex PCR

We applied transcription in vitro following multiplex PCR in order to improve the sensitivity and rapidity of microbial detection in aquatic environment with an oligonucleotide microarray. Transcripts of 16S rRNA gene were fluorescently labeled and hybridized on fabricated oligonucleotide chip. The assay sensitivity was evaluated by detecting cultured bacteria inoculated into natural rive rw ater. By using our procedure, the assay was completed more rapidly (6hr) than conventional oligonucleotide microarray assays (>12hr), and its sensitivity was improved: detection limit was decreased by one order of magnitude. This method might be useful for monitoring pathogenic bacteria in aquatic environments.