Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(?) assays and pyrosequencing, while quantitative, cannot detect less than ～1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(?) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(?) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.     

Rich-defect carbon nanotubes for highly sensitive detection of Dopamine

Dopamine (DA) plays an essential role in the central nervous, renal, hormonal and cardiovascular systems. Various modified carbon nanotubes (CNT)-based dopamine sensors have been reported, but inexpensive, highly sensitive plain CNT-based ones are seldom studied. In this work, a facile and inexpensive CNT-based DA sensor is made by rich-defect multi-walled carbon nanotubes (RD-CNT) via an ultrasound method. The defect and elemental states of the RD-CNT are systematically studied by transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HR-TEM), Raman spectroscopy, X-ray powder diffraction (XRD) and X-ray-photoelectron spectroscopy (XPS). Results show that massive holes and cracks exist in RD-CNT. The level of defects increases from the additional exposed edges. The electrochemical characterizations indicate that the electrochemical sensor has the highest sensitivity of 438.4 μA/(μM ⋅ cm²) among all carbon materials-based DA sensors while well meeting the clinically required detection range and selectivity. The DA sensor was further used to detect live healthy human serum and live PC12 cells with satisfactory results, thus holding great promise for an inexpensive but sensitive DA sensor in practical applications of clinical diagnosis and biological research.     

Metastable state nanoparticle-enhanced Raman spectroscopy for highly sensitive detection

Metastable state silver nanoparticle surface-enhanced Raman scattering has been experimentally and theoretically demonstrated; the signal is two to three orders of magnitude higher than that for the traditional method. Ultrasensitive surface-enhanced Raman scattering signals of illicit drug cocaine and organophosphate pesticide methyl-parathion were observed.     

Contactless conductivity detection for microfluidics: designs and applications

Different methods for construction of contactless conductivity detectors (CCD) for microchip electrophoresis device are described in this review. This includes three main schemes of CCD for microchips, such as (i) the detection electrodes are placed along the microchannel from outside of the microchip and they are insulated from the channel by the cover lid of microchip device; (ii) the electrodes are placed across of the microchannel in the same plane and they are insulated by thin separation channel walls and (iii) electrodes are buried in widened part of microchannel and they are insulated from solution by ultrathin layer of silicon carbide. Specific issues related to the CCD on microfluidics are discussed, such as an influence of shape and magnitude of ac voltage and placement of electrodes and their insulation. Various applications for security, pharmacological, bioassays and food analysis purposes are described.     

Photo-actuation of liquids for light-driven microfluidics: state of the art and perspectives

Using light to control liquid motion is a new paradigm for the actuation of microfluidic systems. We review here the different principles and strategies to induce or control liquid motion using light, which includes the use of radiation pressure, optical tweezers, light-induced wettability gradients, the thermocapillary effect, photosensitive surfactants, the chromocapillary effect, optoelectrowetting, photocontrolled electroosmotic flows and optical dielectrophoresis. We analyze the performance of these approaches to control using light many kinds of microfluidic operations involving discrete pL- to μL-sized droplets (generation, driving, mixing, reaction, sorting) or fluid flows in microchannels (valve operation, injection, pumping, flow rate control). We show that a complete toolbox is now available to control microfluidic systems by light. We finally discuss the perspectives of digital optofluidics as well as microfluidics based on all optical fluidic chips and optically reconfigurable devices.     

Reversible fluorescent probe for highly selective and sensitive detection of mercapto biomolecules

A coumarin-derived complex, Hg(2)L(2), was reported as a highly sensitive and selective probe for the detection of mercapto biomolecules in aqueous solution. The addition of Cys to a 99% aqueous solution of Hg(2)L(2) resulted in rapid and remarkable fluorescence OFF-ON (emission at 525 nm) due to the ligand-exchange reaction of Cys with L coordinated to Hg(2+). The increased fluorescence can be completely quenched by Hg(2+) and recovered again by the subsequent addition of Cys. Such a fluorescence OFF-ON circle can be repeated at least 10 times by the alterative addition of Cys and Hg(2+) to the solution of Hg(2)L(2), indicating that it can be used as a convertible and reversible probe for the detection of Cys. The interconversion of Hg(2)L(2) and L via the decomplexation/complexation by the modulation of Cys/Hg(2+) was definitely verified from their crystal structures. Other competitive amino acids without a thiol group cannot induce any fluorescence changes, implying that Hg(2)L(2) can selectively determine mercapto biomolecules. Using confocal fluorescence imaging, L/Hg(2)L(2) as a pair of reversible probes can be further applied to track and monitor the self-detoxification process of Hg(2+) ions in SYS5 cells.     

Dual-site fluorescent probe for highly selective and sensitive detection of sulfite and biothiols

A dual-site fluorescent probe that could discriminatively respond to Cys and HSO^3- through two emission channels was reported, and it could further applied in imaging biothiols in living cells.     

Carbon nanospheres for highly sensitive electrochemical detection of sequence-specific protein-DNA interactions

A carbon nanosphere-based electrochemical detection method is developed for the highly sensitive detection of Sp1-DNA interactions.     

Facile synthesis of two-dimensional PA-Ag@C film for highly sensitive SERS detection

In this study, the surface of polyamide (PA) films are electrostatically deposited with the carbon-coated silver (Ag@C) nanoparticles, resulting in a two-dimensional (2D) PA-Ag@C film substrate. The TEM images demonstrate that the nanoparticles were successful synthesized. By adjusting the pH of the system, the core–shell structure and the 2D SERS substrate work together to improve the sensitivity, stability, and repeatability of the substrate to be used in complex real-world water samples. The SERS enhancement effect and substrate uniformity were determined using rhodamine 6G (R6G), crystal violet (CV), and malachite green (MG). The results indicate that the 2D PA-Ag@C film substrate in this study has the optimal Raman effect at a system pH of 6. Under ideal pH conditions, the R6G detection limit (LOD) is as low as 10⁻¹⁰ M (D2 attenuation), and the Raman signal intensity deviation of the same substrate is maintained within 9.49%. Overall, the Raman signal of probe molecule on the fabricated PA-Ag@C film possesses excellent sensitivity, repeatability, and stability.     

A highly sensitive fluorogenic chemodosimeter for rapid visual detection of phosgene

A highly sensitive chemodosimeter was identified from a panel of rhodamine derivatives for rapid and visual detection of phosgene with a detection limit of 50 nM triphosgene. Visual detection of gaseous phosgene with chemodosimeter absorbed paper strips was demonstrated.     

Electrochemical detection of hydrazine using a highly sensitive nanoporous gold electrode

A facile alloy–dealloy technique performed in aqueous media was employed to prepare a nanoporous gold (NPG) electrode that demonstrated extremely high sensitivity toward hydrazine oxidation. An Ag_∼60Au_∼40 alloy was electrodeposited at a constant potential on sequentially Cr- and Au-deposited indium tin oxide (Au/Cr/ITO) from a bath that contained sulfuric acid, thiourea, HAuCl₄·3H₂O, and AgNO₃. The dealloying step was performed in concentrated HNO₃, where Ag in the alloy was selectively oxidized to leave the NPG structure. The NPG electrode was employed to study the hydrazine oxidation in basic phosphate buffer solution (PBS), and the results were compared with those obtained using the gold nanoparticle (AuNP)-modified ITO (AuNP/ITO) electrode. The NPG electrode demonstrated an unusual surface-confined behavior, which probably resulted from the thin-layer characteristics of the nano-pores. Hydrazine was detected by hydrodynamic chronoamperometry (HCA) at +0.2 V (vs. Ag/AgCl). The steady-state oxidative current exhibited a linear dependence on the hydrazine concentration in the concentration range of 5.00 nM–2.05 mM, and the detection limit was 4.37 nM (σ = 3). This detection limit is the lower than the detection limits reported in the current literature concerning the electrochemical detection of hydrazine. The NPG electrode indeed demonstrates greater stability after hydrazine detection than the AuNP/ITO electrode.     

Two- and three-wavelength laser multiphoton ionization for highly sensitive detection in solution

Novel two- and three-wavelength laser multiphoton ionization techniques for highly sensitive detection in solution have been established. The photocurrent signal obtained for benzo[a]pyrene by irradiation at 355 nm in n-heptane was effectively enhanced by additional simultaneous irradiation at 532 and/or 1064 nm. The additional irradiation at 532 nm (5 mJ) doubled the signal-to-noise ratio, while that at 1064 nm (30 mJ) increased it 5.5-fold relative to that obtained when only the 355 nm radiation was used. The simultaneous action of 355, 532 (5 mJ) and 1064 (25 mJ) nm radiation further improved the S/N ratio; the detection limit was as low as 1.9 x 10(-10)M. The 532 nm radiation enhanced the photocurrent signal more effectively than did the 1064 nm radiation.     

Streptavidin-enhanced surface plasmon resonance biosensor for highly sensitive and specific detection of microRNA

We are presenting a method for sensitive and specific detection of microRNA (miRNA) using surface plasmon resonance. A thiolated capture DNA probe with a short complete complementary sequence was immobilized on the gold surface of the sensor to recognize the part sequence of target miRNA, and then an oligonucleotide probe linked to streptavidin was employed to bind the another section of the target. The use of the streptavidin-oligonucleotide complex caused a ~5-fold increase in signal, improved the detection sensitivity by a factor of ~24, and lowered the detection limit to 1.7 fmol of miR-122. This specificity allowed a single mismatch in the target miRNA to be discriminated. The whole assay takes 30 min, and the surface of the sensor can be regenerated at least 30 times without loss in performance. The method was successfully applied to the determination of miRNA spiked into human total RNA samples.

Biosensors based on highly sensitive acetylcholinesterases for enhanced carbamate insecticides detection

This paper presents the construction of amperometric biosensors for the highly sensitive detection of carbamate insecticides based on the inhibition of acetylcholinesterase (AChE). This enzyme was immobilised by entrapment in an optimised sol-gel matrix on TCNQ-modified screen-printed electrodes. The enzyme activity was estimated by measuring the thiocholine produced by the enzymatic hydrolysis of the acetylthiocholine using TCNQ as mediator. Wild and genetically engineered AChEs from Drosophila melanogaster (Dm) were chosen for their high sensitivity towards insecticides, which substantially improves the LOD compared with cholinesterases from other sources. The wild type and three mutant enzymes were tested against three carbamate insecticides: carbaryl, carbofuran and pirimicard. The best LOD were obtained with the Y370A mutant for carbaryl (1 × 10⁻⁸ M), the E69W mutant for pirimicarb (2 × 10⁻⁸ M) and the I161V mutant for carbofuran (8 × 10⁻¹⁰ M). The biosensors were applied to the analysis of two potable water samples.     

Gold nanoparticle aggregation-based highly sensitive DNA detection using atomic force microscopy

The potential ability of atomic force microscopy (AFM) as a quantitative bioanalysis tool is demonstrated by using gold nanoparticles as a size enhancer in a DNA hybridization reaction. Two sets of probe DNA were functionalized on gold nanoparticles and sandwich hybridization occurred between two probe DNAs and target DNA, resulting in aggregation of the nanoparticles. At high concentrations of target DNA in the range from 100 nM to 10 μM, the aggregation of gold nanoparticles was determined by monitoring the color change with UV-vis spectroscopy. The absorption spectra broadened after the exposure of DNA–gold nanoparticles to target DNA and a new absorption band at wavelengths >600 nm was observed. However, no differences were observed in the absorption spectra of the gold nanoparticles at low concentrations of target DNA (10 pM to 10 nM) due to insufficient aggregation. AFM was used as a biosensing tool over this range of target DNA concentrations in order to monitor the aggregation of gold nanoparticles and to quantify the concentration of target DNA. Based on the AFM images, we successfully evaluated particle number and size at low concentrations of target DNA. The calibration curve obtained when mean particle aggregate diameter was plotted against concentration of target DNA showed good linearity over the range 10 pM to 10 nM, the working range for quantitative target DNA analysis. This AFM-based DNA detection technique was three orders of magnitude more sensitive than a DNA detection method based on UV-vis spectroscopy.     

Aptamer-based highly sensitive electrochemical detection of thrombin via the amplification of graphene

Herein, we successfully fabricated a highly sensitive label-free electrochemical aptasensor for thrombin based on the amplification of graphene (Gra). The excellent electrochemical probe of nickel hexacyanoferrate nanoparticles (NiHCFNPs) was introduced to form Nafion-Graphene-NiHCFNPs (Nf-Gra-NiHCFNPs) nanocomposites membrane on the gold electrode. The employment of graphene not only enhanced the surface area of the electrode with increased NiHCFNPs immobilization, but also improved the conductivity of the electrode, which further effectively improved the sensitivity of this proposed aptasensor. Subsequently, AuNPs layer was formed to immobilize the thrombin aptamer (TBA) and enhance the stability of the composite monolayer mentioned above. Then, thiol-modified TBA was assembled onto the AuNPs layer. Thereafter, hexanethiol (HT) was employed to block the possible remaining active sites. With the dual amplification of Gra and AuNPs, the resulting aptasensor exhibited good current response to target thrombin with a wide linear range extended from 1 pM to 80 nM (the detection limit was 0.3 pM). Additionally, the morphologies of bare Au substrate, nickel hexacyanoferrate nanoparticles (NiHCFNPs) and nanocomposites were successfully characterized by atomic force microscopy (AFM).     

A simple fluorescent sensor for highly sensitive detection of UO22+

Extensive application of nuclear energy has caused widespread environmental uranium contamination. New detection approaches without complicated sample pretreatment and precision instruments are in demand for on-site and in-time determination of uranyl ions in environmental monitoring, especially in an emergency situation. In this work, a simple and effective fluorescent sensor (Z)-N'-hydroxy-4-(1,2,2-triphenylvinyl)benzimidamide (TPE-A) with aggregation-induced emission (AIE) character was established and studied. It could realize to detect UO₂²⁺ via quenching the fluorescence of its aggregation-induced emission, with good selectivity and sensitivity. Such strategy shows a wide linear range from 5.0 × 10^?8 mol/L to 4.5 × 10^?7 mol/L (R² = 0.9988) with exceptional sensitivity reaching 4.7 × 10^?9 mol/L, which is far below the limit for uranium in drinking water (30 μg/L, ca. 1.1 × 10^?7 mol/L) stipulated by the WHO. A response time less than four minutes make it rapid for uranyl ion measurement. It was applied for detection of uranyl ion in spiked river water samples with recoveries in the range of 98.7%-104.0%, comparable to those obtained by ICP-MS. With the advantages of portable apparatus, rapid detection process and high sensitivity, TPE-A can serve as a promising fluorescent sensor for the detection of UO₂²⁺ in environmental water samples.     

Novel facile detection of persistent organic pollutants using highly sensitive gas sensor

Persistent organic pollutants (POPs) are greatly noxious chemicals in environment, and they can cumulate in organisms and transfer between different species. Therefore, it is significant to detect POPs for both environmental evaluation and further treatment. However, developing facile approach for the detection of POPs still remains a challenge so far. In this paper, we report an innovative method for facile detection of POPs using gas sensor for the first time. Porous SnO₂ nanostructures with a special tri-walled structure prepared via hydrothermal route and annealing process, were employed as gas-sensing materials. Through gas measurements, it was revealed that the as-fabricated gas sensor exhibited highly sensitive performance towards target POPs, including methoxychlor, mirex, p,p′-DDT, and aldrin. Moreover, we found that target POPs were distinguishable by extracting characteristics in kinetic curves of gas adsorption-desorption. As the presented detecting approach is facile without the requirements of complex operations, expensive and bulky instruments, it is expected that it would be developed as a promising method for the detection of POPs, and thereby showing its significance for environmental monitoring.