Ultrasensitive electrical biosensing of proteins and DNA: carbon-nanotube derived amplification of the recognition and transduction events

A new strategy for dramatically amplifying enzyme-linked electrical detection of proteins and DNA using carbon nanotubes (CNTs) for carrying numerous enzyme tracers and accumulating the enzymatically liberated product on CNT-modified transducer is described. Such a CNT-derived double-step amplification pathway (of both the recognition and transduction events) allows the detection of DNA and proteins down to 1.3 and 160 zmol, respectively, in 25-50 muL samples and indicates great promise for PCR-free DNA analysis. The new protocol is illustrated for monitoring sandwich hybridization and antibody-antigen interactions in connection with alkaline-phosphatase tracers. The DNA-linking of CNTs and particles holds promise also for assembling hybrid nanostructures relevant to molecular electronic devices.     

Direct detection of genomic DNA by enzymatically amplified SPR imaging measurements of RNA microarrays

A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.     

Ultrasensitive and selective DNA detection by hydroxylamine assisted gold nanoparticle amplification

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization which can achieve ultra-sensitivity up to approximately 30 zmol, i.e. 300 aM.     

Fast and sensitive detection of ochratoxin A in red wine by nanoparticle-enhanced SPR

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL⁻¹ and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL⁻¹ by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied.     

Upconversion emission of fluorescent silver nanoclusters and in situ selective DNA biosensing

Due to their good photostability, high quantum yield and low toxicity, fluorescent silver nanoclusters (Ag NCs) have received much attention as novel fluorophores for sensing applications. In this work, we investigate the upconversion emission of Ag NCs templated by single- and double-stranded DNAs. DNA-templated Ag NCs exhibit upconversion emission at wavelengths identical to those observed for the corresponding Stokes emission. Consequently, the Ag NCs' upconversion behavior can be easily tuned by the used DNA sequences. In addition, the Ag NCs are more stable under such an NIR excitation with the upconversion mode relative to the Stokes mode. As a proof-of-concept application, DNA nucleobase recognition with the in situ formed Ag NCs is realized using the Ag NCs' upconversion emission. We expect that the Ag NCs' upconversion emission is more advantageous than the previously used rare-earth materials, at least with respect to easy modulation of the emission energies by DNA sequences, and could find wide application in sensor design.     

Photoelectrochemical determination of Hg(II) via dual signal amplification involving SPR enhancement and a folding-based DNA probe

The authors describe a highly sensitive and selective photoelectrochemical (PEC) assay for mercury(II) ions. It is based on a dual signal amplification strategy. The first enhancement results from the surface plasmon resonance (SPR) of Au@Ag nanoparticles (NPs) absorbed on MoS₂ nanosheets. Here, the injection of hot electrons of Au@Ag NPs into MoS₂ nanosheets produces a strong photocurrent, while background signals are strongly reduced. The second enhancement results from the use of a thymine rich ct-DNA aptamer attached to the Au@Ag-MoS₂ nanohybrid. The DNA specifically binds Hg(II) ions to form thymine-Hg(II)-thymine (T-Hg-T) complexes. This leads to the formation of a hairpin-shaped dsDNA structure. The use of a CdSe quantum dot label at the terminal end of the ct-DNA further facilitates electron–hole separation. The photocurrent of the detector is measured as a function of Hg(II) concentration at a bias voltage of 0.1 V and under irradiation of 430 nm light. Due to the two-fold amplification strategy presented here, the linear range extends from 10 pmol·L^?1 to 100 nmol·L^?1, with a detection limit of 5 pmol·L^?1 (at S/N?=?3).

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Graphical Abstract The injection of hot electrons of Au@Ag into MoS₂ produces a strong photocurrent, and the formation of thymine-Hg(II)-thymine further facilitates electron–hole separation by CdSe. This dual signal amplification strategy is used to detect Hg(II) ions via a photoelectrochemical assay.

     

DNA microarray fabricated on poly(acrylic acid) brushes-coated porous silicon by in situ rolling circle amplification

Microarrays hold considerable promise in large-scale biology on account of their analytical, massive and parallel nature. In a step toward further enabling such a capability, we describe the application of rolling circle amplification (RCA) for a sensitive and multiplex detection of nucleic acid targets on oligonucleotide-conjugated polymer brushes covalently grown from porous silicon. Both RCA and polymer brushes have been taken to increase the loading quantity of target molecules and thus improve the detection sensitivity without loss of multiplexing. Besides, polymer brushes were employed to protect porous silicon and to provide biologically simulated environments, making the attached biomolecules maintain bioactivity. This approach can reach a detection limit of 0.1 nM target analytes and three orders of magnitude dynamic range of 0.1-100 nM, with a fluorescence scanner. A two-colour DNA microarray was achieved via RCA of two kinds of circular DNA targets on one chip simultaneously. The porous silicon chip-based RCA technique is promising for the multiplex detection of deoxynucleic acids on microarrays.     

Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray

Aberrant DNA methylation of CpG sites has been confirmed to be closely associated with carcinogenesis.Based on the hyperbranched rolling circle amplification(HRCA) and microarray techniques,a new method for qualitative detection of methylation was developed.In the present study,padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes.Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction.Utilizing the method of 3-dimensional polyacrylamide gel-based microarray,the HRCA product will be immobilized on the slide to form a DNA microarray,which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites.To control the false positive signals,DNA ligase and temperature of ligation/digestion are optimized.Methylation status of four CpG sites located in P15,Ecadherin,hMLH1 and MGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR.Our research proves that this method is simple and inexpensive,and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.     

Ultrasensitive fluorescence polarization DNA detection by target assisted exonuclease III-catalyzed signal amplification

Single stranded DNA sequences can be detected by target assisted exonuclease III-catalyzed signal amplification fluorescence polarization (TAECA-FP). The method offers an impressive detection limit of 83 aM within one hour for DNA detection and exhibits high discrimination ability even against a single base mismatch.     

Ultrasensitive DNA detection based on Au nanoparticles and isothermal circular double-assisted electrochemiluminescence signal amplification

We report here an ultrasensitive DNA detection approach which combines Au NPs enhanced electrochemiluminescence (ECL) of the CdS nanocrystal (NC) film with isothermal circular amplification reaction of polymerase and nicking endonuclease (NEase). By the double-signal amplification, this approach could sensitively respond down to 5 aM DNA.     

Advances in signal amplification strategies for electrochemical biosensing

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SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes

Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.     

DNA detection and signal amplification via an engineered allosteric enzyme

Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 fmol DNA in less than 3 min under physiological conditions.     

Recent progress in gold nanoparticle-based biosensing and cellular imaging

Gold nanoparticles (AuNPs) have been extensively used in optical biosensing and bioimaging due to the unique optical properties. Biological applications including biosensing and cellular imaging based on optical properties of AuNPs will be reviewed in the paper. The content will focus on detection principles, advantages and challenges of these approaches as well as recent advances in this field.     

Emerging advances in plasmonic nanoassemblies for biosensing and cell imaging

Integrating discrete plasmonic nanoparticles into assemblies can induce plasmonic coupling that produces collective plasmonic properties, which are not available for single nanoparticles. Theoretical analysis revealed that plasmonic coupling derived from assemblies could produce stronger electromagnetic field enhancement effects. Thus, plasmonic assemblies enable better performance in plasmon-based applications,such as enhanced fluorescence and Raman effects. This makes them hold great potential...     

Quantitative angle-resolved SPR imaging of DNA-DNA and DNA-drug kinetics

We demonstrate the quantitative characterization of DNA-DNA and DNA-drug interactions by angle-resolved surface plasmon resonance (SPR) imaging. Combining the angle-scanning capabilities of traditional SPR with the spatial definition capabilities of imaging, we directly measure DNA and drug surface coverages and kinetics simultaneously for multiple patterned spots. We find excellent agreement of DNA-DNA hybridization kinetics and thermodynamics measured by both the imaging system and traditional SPR. Instrument response and sensitivity is further demonstrated by successful measurement of association and dissociation kinetics of actinomycin-D binding to a low-density doubled-stranded DNA binding sequence. Without independent calibration, analysis of angle-resolved SPR imaging data yields 2.9 +/- 0.1 drugs per duplex at saturation coverage, consistent with all available duplex binding sites being occupied.     

Rapid microarray detection of DNA and proteins in microliter volumes with surface plasmon resonance imaging measurements

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm(2) area; 45 nm thickness), each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18?000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.     

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo⁻), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.     

External stimulation-controlled dynamic DNA devices for biosensing and biomedical applications

DNA devices that can recognize molecular inputs and transform them into functional outputs in an autonomous manner have been actively pursued as versatile toolkits for controlled nanofabrication, molecular network regulation, biosensing and cellular function modulation. The introduction of external stimuli-responsive units not only ensures the programmability and functionality of DNA devices themselves, but also confers rapid, remote and reversible dynamic regulation capabilities. This facilitat...