A label-free and signal-on supersandwich fluorescent platform for Hg sensing

A label-free supersandwich fluorescent assay was demonstrated for the first time by taking Hg²⁺ as a detection candidate. The principle of the proposed supersandwich fluorescent platform is based on the formation of supersandwich structure by T-Hg²⁺-T coordination and the fluorescence enhancement of the intercalated Genefinder (GF) in double strand DNA (dsDNA). Such supersandwich fluorescent DNA sensor exhibits a linear range of 10–300 nM for the detection of Hg²⁺, with a detection limit of 2.5 nM on the basis of the 3σ/slope (σ represents the standard deviation of the blank samples), which is well below the permit of the U.S. Environmental Protection Agency (<10 nM). The detection can be fulfilled in less than 10 min. The proposed mix-and-detect fluorescent platform exhibits excellent sensitivity, selectivity, and convenient manipulation. The assay was successfully used to detect Hg²⁺ in the lake water samples, which suggested its potential in practical samples.     

Tin oxide nanoparticles-polymer modified single-use sensors for electrochemical monitoring of label-free DNA hybridization

In this study, SnO₂ nanoparticles (SNPs)-poly(vinylferrocenium) (PVF⁺) modified single-use graphite electrodes were developed for electrochemical monitoring of DNA hybridization. The surfaces of polymer modified and polymer-SNP modified pencil graphite electrodes (PGEs) were firstly characterized by using SEM analysis. The electrochemical behaviours of these electrodes were also investigated using the differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The polymer-SNP modified PGEs were then tested for the electrochemical sensing of DNA based on the changes at the guanine oxidation signals. Experimental parameters, such as; different modifications in DNA oligonucleotides, DNA probe concentrations were examined to obtain more sensitive and selective electrochemical signals for nucleic acid hybridization. After optimization studies, DNA hybridization was investigated in the case of complementary of hepatitis B virus (HBV) probe, mismatch (MM), and noncomplementary (NC) sequences.     

A versatile label-free and signal-on electrochemical biosensing platform based on triplex-forming oligonucleotide probe

Nucleic acid and protein assays are very important in modern life sciences, and the recently developed triplex-forming oligonucleotide probes provide a unique means for biological analysis of different kinds of analytes. Herein, we report a label-free and signal-on electrochemical sensor for the detection of specific targets, which is based on the triple-helix structure formation between the hairpin molecular beacon and the capture probe through the intermolecular DNA hybridization induced by Watson-Crick and Hoogsteen base pairings. Upon the introduction of a specific target, the triple-helical stem region is dissembled to liberate the hemin aptamer, and a G-quadruplex− hemin complex can be formed in the presence of K⁺ and hemin on the electrode surface to give an electrochemical response, thus signaling the presence of the target. With the use of Human Immunodeficiency Virus type 1 (HIV-1) as a proof-of-principle analyte, we first demonstrated this approach by using a molecular beacon, which consists of a central section with the DNA sequence complementary to HIV-1, flanked by two arm segments. This newly designed protocol provides an ultrasensitive electrochemical detection of HIV-1 with a limit of detection down to 0.054 nM, and also exhibit good selectivity. Therefore, the as-proposed strategy holds a great potential for early diagnosis in gene-related diseases, and with further development, it could be used as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.     

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G_NP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G_NP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G_NP were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3−/4− as the redox indicator. The (CA-G_NP/SPAN)_n films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G_NP) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)₆]^3−/4− in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G_NP/SPAN)_n films, the target DNA with 20-base could be detected up to 2.13 × 10⁻¹³ mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.     

Droplet-based microscale colorimetric biosensor for multiplexed DNA analysis via a graphene nanoprobe

The development of simple and inexpensive DNA detection strategy is very significant for droplet-based microfluidic system. Here, a droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device by using fluorescence-based colorimetric method and a graphene nanoprobe. With the aid of droplet manipulation technique, droplet size adjustment, droplet fusion and droplet trap are realized accurately and precisely. Due to the high quenching efficiency of graphene oxide (GO), in the absence of target DNAs, the droplet containing two single-stranded DNA probes and GO shows dark color, in which the DNA probes are labeled carboxy fluorescein (FAM) and 6-carboxy-X-rhodamine (ROX), respectively. The droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of 9.46 and 9.67 × 10⁻⁸ M, respectively. It is also demonstrated that this biosensor platform can become a promising detection tool in high throughput applications with low consumption of reagents. Moreover, the incorporation of graphene nanoprobe and droplet technique can drive the biosensor field one more step to some extent.     

Protein determination using graphene oxide-aptamer modified gold nanoparticles in combination with Tween 80

Recently, graphene oxide (GO) has shown superiority for disease detection arising from its unique physical and chemical properties. However, proteins adsorbed on the surface of GO prevent sensitivity improvement in fluorescence-based detection methods. In this paper, a label-free method based on aptamer modified gold nanoparticles (GNPs) combined with Tween 80 was shown to solve this problem using the detection of thrombin as an example. An aptamer was designed and bound to thrombin by changing its conformation. Tween 80 was used for rapid and reproducible synthesis of stable DNA-functionalized GNPs and prevented the thrombin from nonspecific binding to GO. Thrombin was detected with a limit of 0.68 pM by taking advantage of the efficient cross-linking effect of aptamer-GNPs to GO. The sensor was validated by determining thrombin concentration in human blood serum samples. The results indicate that this method has promising analytical application in medical diagnostic.     

Nanocomposites of gold nanoparticles and graphene oxide towards an stable label-free electrochemical immunosensor for detection of cardiac marker troponin-I

A stable label-free amperometric immunosensor is presented based on gold nanoparticles and graphene oxide nanocomposites for detection of cardiac troponin-I in the early diagnosis of myocardial infarction. For designing of the sensing platform, firstly the nanocomposites based on GO and AuNPs were prepared and anchored on electrode surfaces. The formed nanocomposites provided a platform with big surface area for loading anti-cTnI capture antibody, and worked as a bridge for fast electron transfer subsequently increased the sensitivity. Moreover, the linkages between AuNP, GO, and electrodes were based on covalent bonding by aryldiazonium salt coupling chemistry, which favors the stability of the sensing interface. Finally, the anti-cTnI detection antibody was immobilized on GO tailored with ferrocene molecules, functioning as the signal reporter for the detection of cTnI. The modification process was monitored using electrochemistry, SEM, XPS. The herein immunosensor demonstrates a good selectivity and high sensitivity against human-cTnI, and is capable of detecting cTnI at concentrations as low as 0.05 ng mL⁻¹, which is 100 times lower than that possible by conventional methods. It is potential to design the portable sensing platform based on AuNPs and GO nanocomposites for future point-of-care diagnostics.     

An electrochemical ascorbic acid sensor based on palladium nanoparticles supported on graphene oxide

In this study, an electrochemical ascorbic acid (AA) sensor was constructed based on a glassy carbon electrode modified with palladium nanoparticles supported on graphene oxide (PdNPs-GO). PdNPs with a mean diameter of 2.6 nm were homogeneously deposited on GO sheets by the redox reaction between PdCl₄²⁻ and GO. Cyclic voltammetry and amperometric methods were used to evaluate the electrocatalytic activity towards the oxidation of AA in neutral media. Compared to a bare GC or a Pd electrode, the anodic peak potential of AA (0.006 V) at PdNPs-GO modified electrode was shifted negatively, and the large anodic peak potential separation (0.172 V) of AA and dopamine (DA), which could contribute to the synergistic effect of GO and PdNPs, was investigated. A further amperometric experiment proved that the proposed sensor was capable of sensitive and selective sensing of AA even in the presence of DA and uric acid. The modified electrode exhibited a rapid response to AA within 5 s and the amperometric signal showed a good linear correlation to AA concentration in a broad range from 20 μM to 2.28 mM with a correlation coefficient of R = 0.9991. Moreover, the proposed sensor was applied to the determination of AA in vitamin C tablet samples. The satisfactory results obtained indicated that the proposed sensor was promising for the development of novel electrochemical sensing for AA determination.     

A label-free colorimetric platform for DNA via target-catalyzed hairpin assembly and the peroxidase-like catalytic of graphene/Au-NPs hybrids

A target-catalyzed hairpin assembly (CHA) and graphene/Au-NPs hybrids-based platform has been developed for the determination of DNA. This new sensor not only avoided any labeling but also reduced the background signal. In the absence of target, the assembly of H1 and H2 couldn't be triggered. The catalytic activity of graphene/Au-NPs hybrids was inhibited by adsorption of H1 and H2, leading to the “inactive” hybrids unable to catalyze the oxidation reaction of 3,3′,5,5′-tetramethylbenzidine (TMB). However, with the addition of target DNA, the target-catalyzed hairpin assembly was initiated and produced plenty of H1–H2 duplex, which had a weak binding affinity with the graphene/Au-NPs. Thus, the protected interface of graphene/Au-NPs hybrids became active and catalyzed the oxidation reaction of TMB accompanied with a colorless to-blue color change. This approach exhibited good sensitivity and specificity for target DNA with a detection limit of 5.74 × 10⁻¹¹ M, and realized the assay of target DNA in human serum samples. Besides, this sensor could be further expanded to detect viruses or proteins by adapting the corresponding aptamers, showing great potential in biochemical detections.     

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels.     

Controlling wrinkles and assembly patterns in dried graphene oxide films using lyotropic graphene oxide liquid crystals

We fabricated graphene oxide (GO) films on glass substrates by blade coating a lyotropic GO liquid crystal dispersion. Substrate temperature and blading speed were precisely controlled to manipulate the surface morphologies of GO films. The temperature and blade speed influenced the drying rate of film and the amount GO dispersion supplied. By controlling these parameters, film-thickness modulation and three types of surface wrinkle patterns were selectively achieved. We also plotted the wrinkle patterns diagram as functions of the film fabrication conditions. The films exhibited different optical anisotropies depending on wrinkle patterns. GO films with controlled wrinkles can be used as electrodes for supercapacitor applications owing to the large surface areas.     

Simultaneous electrochemical DNA hybridization assay for PAT and FMV 35S gene sequence using quantum dots as labels

An electrochemical method for the simultaneous detection of two different DNA sequences from PAT and FMV 35S gene sequence using CdS and PbS quantum dots （QDs） as labels was described. The QDs were readily functionalized with oligonucleotides as electrochemical DNA probes and selectively hybridized to the complementary sequences immobilized on the microplate. The QDs anchored on the hybrids were dissolved in the solution by the oxidation of HNO3 and further detected by a sensitive differential pulse anodic stripping voltammetric method （DPASV）. The DPASV signals of the oxidation of Cd^2＋ and Pb^2＋ ions present in the solution were different and reflected the identity of corresponding ssDNA targets sequences.     

A novel label-free colorimetric assay for DNA concentration in solution

Optical devices were fabricated by sandwiching a “monolithic” poly(N-isopropylacrylamide-co-N-(3-aminopropyl) methacrylamide hydrochloride) (pNIPAm-co-APMAH) microgel layer between two semitransparent Au layers. These devices, referred to as etalons, exhibit characteristic multipeak reflectance spectra, and the position of the peaks in the spectra primarily depends on the distance between the Au surfaces mediated by the microgel layer thickness. Here, we show that the positively charged microgel layer can collapse in the presence of negatively charged single stranded DNA (ssDNA) due to ssDNA induced microgel crosslinking. The collapse results in a change in the etalon's optical properties, which can be used to detect target DNA in a complex mixture.     

Facile synthesis of cobalt oxide/reduced graphene oxide composites for electrochemical capacitor and sensor applications

Reduced graphene oxide sheets decorated with cobalt oxide nanoparticles (Co₃O₄/rGO) were produced using a hydrothermal method without surfactants. Both the reduction of GO and the formation of Co₃O₄ nanoparticles occurred simultaneously under this condition. At the same current density of 0.5 A g⁻¹, the Co₃O₄/rGO nanocomposites exhibited much a higher specific capacitance (545 F g⁻¹) than that of bare Co₃O₄ (100 F g⁻¹). On the other hand, for the detection of H₂O₂, the peak current of Co₃O₄/rGO was 4 times higher than that of Co₃O₄. Moreover, the resulting composite displayed a low detection limit of 0.62 μM and a high sensitivity of 28,500 μA mM⁻¹cm⁻² for the H₂O₂ sensor. These results suggest that the Co₃O₄/rGO nanocomposite is a promising material for both supercapacitor and non-enzymatic H₂O₂ sensor applications.     

A label-free electrochemical test for DNA-binding activities of tumor suppressor protein p53 using immunoprecipitation at magnetic beads

In this paper we extend the application area of the label-free structure-sensitive electrochemical DNA sensing with mercury-based electrodes which is for the first time used, in combination with immunoprecipitation at magnetic beads (MB), for the probing of DNA interactions with tumor suppressor protein p53. The technique relies on capture of the p53-DNA complexes at MB via anti-p53 antibodies, followed by salt-induced dissociation of linear DNA from the complex and its voltammetric detection. Competitive binding of p53 to various plasmid DNA substrates, including lin or scDNAs with or without a specific target site, can easily be followed by ex situ electrochemical analysis of DNA recovered from the immunoprecipitated complexes. Compared to gel electrophoresis which is usually applied to analyze different plasmid DNA forms and their complexes with proteins, the electrochemical detection is faster and allows simpler quantitation of DNA containing free ends at submicrogram levels. We demonstrate applicability of the proposed technique to monitor different DNA-binding activities of wild type and mutant p53 proteins.     

Dumbbell probe-mediated cascade isothermal amplification: A novel strategy for label-free detection of microRNAs and its application to real sample assay

Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1 zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.     

Introduction of CdO nanoparticles into graphene and graphene oxide nanosheets for increasing adsorption capacity of Cr from wastewater collected from petroleum refinery

Graphene and graphene oxide nanocomposites are promising and fascinating types of nanocomposites because of their fast kinetics, unique affinity for heavy metals, and greater specific area. Initially, in this study, a green, cost-effective and facile method was utilized to prepare G, GO, CdO, G-CdO, and CdO-GO nanocomposites by Azadirachta indica and then analyzed using UV–vis spectroscopy, Fourier-transform spectroscopy, Raman, X-ray diffraction and scanning electron microscope. The synthesized nanocomposites were explored for chromium elimination from wastewater collected from a petroleum refinery. CdO-GO, G-CdO nanocomposites showed remarkable adsorption capability of 699 and 430 mg g^?1 which was higher than G (80 mg g^?1), GO (65 mg g^?1), and CdO (400 mg g^?1). Based on the R² (correlation coefficient) values, the kinetic statistics of Cr (VI) onto the G, GO, CdO, G-CdO, and CdO-GO were effectively obeyed by pseudo-second-order than by all other models. The R² values for the five nano-bioadsorbents were extraordinarily high (R² greater than 0.990) which ensured the chemisorption. This study ensured that the adsorptive removal rate of Cr (VI) is still greater than 85 % after repeated five cycles, suggesting that the produced nanomaterials are adsorbents with strong recyclability.     

Toehold-mediated nonenzymatic amplification circuit on graphene oxide fluorescence switching platform for sensitive and homogeneous microRNA detection

A novel graphene oxide (GO) fluorescence switch-based homogenous system has been developed to solve two problems that are commonly encountered in conventional GO-based biosensors. First, with the assistance of toehold-mediated nonenzymatic amplification (TMNA), the sensitivity of this system greatly surpasses that of previously described GO-based biosensors, which are always limited to the nM range due to the lack of efficient signal amplification. Second, without enzymatic participation in amplification, the unreliability of detection resulting from nonspecific desorption of DNA probes on the GO surface by enzymatic protein can be avoided. Moreover, the interaction mechanism of the double-stranded TMNA products contains several single-stranded toeholds at two ends and GO has also been explored with combinations of atomic force microscopy imaging, zeta potential detection, and fluorescence assays. It has been shown that the hybrids can be anchored to the surface of GO through the end with more unpaired bases, and that the other end, which has weaker interaction with GO, can escape GO adsorption due to the robustness of the central dsDNA structures. We verified this GO fluorescence switch-based detection system by detecting microRNA 21, an overexpressed non-encoding gene in a variety of malignant cells. Rational design of the probes allowed the isothermal nonenzymatic reaction to achieve more than 100-fold amplification efficiency. The detection results showed that our strategy has a detection limit of 10 pM and a detection range of four orders of magnitude.     

Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing

Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody–antigen interaction in the presence of casein kinase II (CK2) and adenosine 5′-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis.