Bio Bar Code Research Articles

An electrochemical DNA biosensor coupled with dual amplification strategy based on DNA-Au bio-bar codes and silver enhancement for highly sensitive and selective detection of MDR1 gene

Given the tumors might develop multidrug resistance (MDR) during chemotherapy that leading to treatment failure, rapid and sensitive determination of MDR1 gene is an effective tool to monitor the development of MDR. Herein, we established an electrochemical deoxyribonucleic acid (DNA) biosensor coupled with dual amplification strategy based on DNA-Au bio-bar codes and enhancement of silver for sensitive and selective determination of MDR1 gene. Specifically, DNA-Au bio-bar codes as signal probe were prepared by immobilizing complementary and non-complementary sequences on gold nanoparticles (AuNPs), so as to reduce the cross reaction between the target and reporter DNA. In addition, AuNPs-catalyzed reduction of silver ions greatly amplified the current signal. Under the optimal conditions, the current signal was proportional to the base-10 logarithm of target DNA concentration in a wide range from 100 fM to 1 nM with a detection limit as low as 33 fM (S/N = 3). Moreover, without the need for target amplification, the prepared biosensor exhibited superior selectivity and high sensitivity even in the presence of other sequences. Therefore, the developed electrochemical DNA biosensor is a facile and sensitive platform for MDR1 gene detection.

Sensitive detection of streptomycin in milk using a hybrid signal enhancement strategy of MOF-based bio-bar code and target recycling

A MOF-based bio-bar code material was synthesized and firstly applied to develop an electrochemical streptomycin (STR) aptasensor. By using MOF-based bio-bar code and enzyme-assisted target recycling for dual-signal amplification, highly sensitive detection of STR was achieved. The sensing surface was simply fabricated by immobilizing a mixed monolayer of thiolated cDNA/aptamer duplexes (dsDNA) and 6-mercapto-1-hexanol (MCH) on the gold nanoparticle modified screen printed carbon electrode (Au/SPCE). The presence of target STR caused highly efficient removal of the aptamers from dsDNA assisted by Exo I enzyme. Then MOF-based bio-bar codes were backfilled to achieve the adsorption of electroactive Ru(NH3)63+ (RuHex) on electrode surface. The electrochemical signal of the surface-confined RuHex was used for quantitation. The analytical performance for STR was satisfactory with a wide linear range of 0.005–150 ng mL−1, a low detection limit of 2.6 pg mL−1 and a good selectivity towards other three antibiotics. Moreover, the application of this aptasensor for determination of STR in real milk samples was also realized. With these merits, this dual-signal amplification assay might provide one of the effective ways for food safety monitoring.

Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6pgmL-1 with a large dynamic range from 10 pgmL-1 to 1 µgmL-1.

Molybdenum disulfide/graphdiyne-based photoactive material derived photoelectrochemical strategy for highly sensitive MicroRNA assay

In this study, molybdenum disulfide-graphdiyne (MoS2-GDY) was successfully prepared. Utilizing the inherent reducibility of GDY, (NH4)2MoS4 was reduced to MoS2 forming photoactive material MoS2-GDY complex. MoS2-GDY made use of the large band-gap of MoS2 and the narrow band-gap of GDY. The naturally narrow band-gap of GDY enhanced the absorption of light and the combination of GDY and MoS2 reduced the electron-hole recombination rate so as to gain strong photoelectrochemical (PEC) signal. The PEC signal of MoS2-GDY changed with the mass ratio of GDY to (NH4)2MoS4. MoS2-GDY produced the strongest PEC performance at 1:25. MoS2-GDY with the mass ratio of 1:25 was used to fabricate a PEC assay for microRNA-141 detection. To obtain low background noise and strong PEC signal, a dual-modified probe coupling bio-bar code AuNPs and hybridization chain reaction strategy was designed. The dual-modified probe which labeled with MoS2-GDY on the one side, and marked with biotin on the other side. And the intensity of PEC depended on the concentration of microRNA-141. Under optimal conditions, 0.2 V potential, pH 7.5, the PEC assay had a linear range in 1.0 × 10−17 mol/L to 1.0 × 10-9 mol/L and a low detection limit of 3.3 × 10-18 mol/L. The assay has been successfully applied to the determination of microRNA-141 in complex biological matrixes.

Bimetallic nanomaterials as nanocatalysts and the carrier coupling chemiluminescence aptamer strategy for cancer cell detection

Cu/Co bimetallic nanomaterials (BNMs) were synthesized firstly. The chemiluminescence (CL) catalytic properties of BNMs were investigated systemically. Experimental results revealed that combined Cu with Co could cause a 67.9 ± 3.5% increase in the CL intensity because of a great synergic catalytic effect. Based on this interesting phenomenon, a steric hindrance strategy for cancer cell detection was fabricated. BNMs were used as nanocatalysts and the carrier modified with capture DNA. In presence of CCRF-CEM cell, sgc8 aptamer and probe DNA recognized with CCRF-CEM. And the bio bar code CCRF-CEM cells were formed. After bio bar code CCRF-CEM cells hybridized with capture DNA which modified Cu/Co BNMs, CL probe was introduced. The CL intensity is linearly related to the concentration of cell from 500 to 100,000 cells/mL. The detection limit is 270 cells/mL and the relative standard deviation is 3.9% at a level of 1000 cells/mL (n = 9). This method was successfully applied to the determination of cell in spiked real human serum. Due to its sensitivity and selectivity, this method offers a promising approach for the detection of cell and other biomolecules.

Dual molecular recognition strategy for highly sensitive electrochemical detection of dopamine based on amplification of DNA–Au bio–bar codes

We had reported an electrochemical assay for dopamine with high sensitivity and selectivity based on dual molecular recognition strategy and amplification of DNA–Au bio–bar codes. In this assay, carbon nanotubes were first immobilized on the glass carbon electrode surface. Subsequently, a boronic acid-functionalized pyrene probe was self-assembled on the sidewalls of carbon nanotubes via π–π stacking interactions as a capture probe. 3,3′-Dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP) and double strands DNA functionalized AuNPs (DSP/AuNPs/dsDNA) were designed as signal probes and characterized by transmission electron microscopy and spectroscopic techniques. Upon the binding of dopamine with boronic acid followed by DSP/AuNPs/dsDNA, the AuNPs were captured on the electrode surface to enhance the signal. RuHex, an electroactive complex, which could bind to the anionic phosphate of DNA strands through electrostatic interaction, serves as signaling transducer. Signal amplification and dual recognition were used to quantify dopamine concentration from 30 pM to 30 nM, with a detection limit of 18 pM. Other small molecules could be measured by this assay after modification with differential recognition molecules.

Controllable fabrication of bio-bar codes for dendritically amplified sensing of human T-lymphotropic viruses.

Human T-lymphotropic virus type II (HTLV-II) is an important type-C retrovirus, closely related to a variety of human diseases. Here, we demonstrate for the first time the controllable fabrication of bio-bar codes for dendritically amplified sensing of low-abundant HTLV-II DNA by the integration of terminal deoxynucleotidyl transferase (TdT)-catalyzed template-free polymerization extension with bio-bar-code amplification (BCA). HTLV-II DNA hybridizes with magnetic microparticle (MMP)-modified capture probe 1, forming a stable DNA duplex with a protruding 3'-hydroxylated sequence which may function as a primer to initiate the TdT-catalyzed first-step polymerization extension for the generation of a poly-thymidine (T) sequence. The resultant poly-T products may hybridize with poly-adenine (A) capture probe 2, inducing the self-assembly of multiple capture probe 2-/reporter probe-functionalized Au nanoparticles (AuNPs) onto the MMP. Subsequently, the reporter probes may act as the primers to initiate the TdT-catalyzed second-step polymerization extension, producing large numbers of G-rich DNAzymes for the generation of an enhanced chemiluminescence signal. Taking advantage of the efficient polymerization extension reaction catalyzed by TdT, the high amplification efficiency of BCA, and the intrinsically high sensitivity of G-rich DNAzyme-driven chemiluminescence, this method exhibits ultrahigh sensitivity with a limit of detection of as low as 0.50 aM and a large dynamic range of 9 orders of magnitude from 1 aM to 1 nM. Moreover, this method can be applied for the discrimination of a single-base mismatch and the measurement of HTLV-II DNA in both human serum and human T-lymphocytic leukemia cells, holding great potential in biomedical research and clinical diagnosis.

Nanoparticle based bio-bar code technology for trace analysis of aflatoxin B1 in Chinese herbs

A novel and sensitive assay for aflatoxin B1 (AFB1) detection has been developed by using bio-bar code assay (BCA). The method that relies on polyclonal antibodies encoded with DNA modified gold nanoparticle (NP) and monoclonal antibodies modified magnetic microparticle (MMP), and subsequent detection of amplified target in the form of bio-bar code using a fluorescent quantitative polymerase chain reaction (FQ-PCR) detection method. First, NP probes encoded with DNA that was unique to AFB1, MMP probes with monoclonal antibodies that bind AFB1 specifically were prepared. Then, the MMP-AFB1-NP sandwich compounds were acquired, dehybridization of the oligonucleotides on the nanoparticle surface allows the determination of the presence of AFB1 by identifying the oligonucleotide sequence released from the NP through FQ-PCR detection. The bio-bar code techniques system for detecting AFB1 was established, and the sensitivity limit was about 10−8 ng/mL, comparable ELISA assays for detecting the same target, it showed that we can detect AFB1 at low attomolar levels with the bio-bar-code amplification approach. This is also the first demonstration of a bio-bar code type assay for the detection of AFB1 in Chinese herbs.

Electrochemical biosensor for silver ions based on amplification of DNA–Au bio–bar codes and silver enhancement

A highly sensitive and selective electrochemical DNA biosensor for detection of Ag+ was developed based on amplification of DNA–Au bio–bar codes and silver enhancement. Cytosine-rich single stranded DNA oligonucleotide (sub–DNA) was immobilized on the surface of the gold electrode via Au–S chemical interaction. In the presence of Ag+, with the interaction of cytosine–Ag+–cytosine (C–Ag+–C), cytosine-rich DNA oligonucleotide probes-labeled AuNPs have a hybridization with sub–DNA and then form a duplex-like structure, following the AuNPs-catalyzed silver enhancement. The whole detection procedure of Ag+ based on the developed biosensor was evaluated by electrochemical techniques. Owing to the hybridization of sandwich assay format and distinct electrochemical signal amplification of silver enhancement, the proposed biosensors exhibited a good analytical performance for sensitive transduction of Ag+ recognition with a wide detection linear range from 5pM to 50μM, and a low detection limit of 2pM. The developed biosensor was highly selective and its practical applicability in real water samples was also tested with satisfactory results.

A bio-inspired sensor coupled with a bio-bar code and hybridization chain reaction for Hg(2+) assay.

In this article, a bio-inspired DNA sensor is developed, which is coupled with a bio-bar code and hybridization chain reaction. This bio-inspired sensor has a high sensitivity toward Hg(2+), and has been used to assay Hg(2+) in the extraction of Bauhinia championi with good satisfaction.

A sensitive electrochemical method for DNA methyltransferase assay and inhibitor screening based on DNA methylation-sensitive cleavage

A sensitive electrochemical method for detection of DNA methylation and assay of DNA methyltransferase (Dam) was investigated based on methylation-sensitive restriction endonuclease, DNA-Au bio bar code and enzymatic signal amplification. After the DNA probe was hybridized with complementary DNA sequence, and the obtained double-strand DNA (dsDNA) was further methylated by Dam MTase, it could be successively digested by Dpn I at the methylated sites. As a result, the bio bar code and streptavidin-horseradish peroxidase (S-HRP) could not be conjugated on the electrode surface through the specific interaction between biotin and S-HRP, where biotin was labeled at the DNA-Au bio bar code. Thus, only a low electrochemical reduction signal was obtained. However, the hybridized probe could hybrid with DNA-Au bio bar code, and then S-HRP could be conjugated on the electrode surface in the absence of Dam MTase and Dpn I, so a larger reduction current of enzymatic product was obtained. The change of the reduction current of enzymatic product was used to detect DNA methylation and Dam MTase activity. The detection limit of Dam MTase was 0.02unit/mL (S/N=3). Moreover, the inhibitions of mitomycin C, paclitaxel, chlorogenic acid and epicatechin were also investigated. Therefore, this method provided a sensitive platform for monitoring DNA methylation and DNA MTase activity.

DNAzyme-guided polymerization of aniline for ultrasensitive electrochemical detection of nucleic acid with bio-bar codes-initiated rolling circle amplification

A novel and versatile amplified electrochemical strategy for ultrasensitive detection of nucleic acid has been developed on the basis of polyaniline (PANI) as the redox label which was guided and deposited by multiple HRP-mimicking DNAzyme as a powerful catalyst and template generated by bio-bar codes-initiated rolling circle amplification (RCA). The “sandwich-type” DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe that on the bio-bar codes, respectively. After the RCA reaction, multiple HRP-mimicking DNAzyme were successfully formed, which subsequently catalyzed the oxidation of aniline to PANI and lead to a readily measurable “turn-on” electrochemical signal. As a result of multiple amplification effects, the constructed platform exhibited a good linear response toward nucleic acid over a wide range of concentration from 6.0fM to 1.0nM.

Electrochemical determination of microRNA-21 based on bio bar code and hemin/G-quadruplet DNAenzyme

A simple and novel microRNA (miRNA) biosensor was developed using DNA-Au bio bar code (DNA-Au) and G-quadruplex-based DNAenzyme. DNA-Au increased the amount of miRNA-21 participating in hybridization. Hemin/G-quadruplex DNAenzyme significantly improved the catalysis of H(2)O(2) by oxidation of hydroquinone, resulting in an obvious reduction current of benzoquinone for miRNA-21 indirect detection. Under optimum conditions, the linear relationship between miRNA-21 concentration and reduction response was obtained with the detection limit of 0.006 pM, which showed a good sensitivity. Besides, selectivity of the biosensor was investigated by detecting the base mismatched miRNAs. This proposed method was further applied to detect miRNA-21 extracted from human hepatocarcinoma BEL-7402 cells and human mastocarcinoma MCF-7 cells. The influence of bisphenol A (BPA) on the expression of miRNA-21 in cells was also investigated. The biosensor performs well in practical applications, which suggests it may provide a new platform for gene diagnosis.

Ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence method for the detection of protein

An ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence (IM-ECL) method for the detection of protein is developed. The target protein is captured by biotin-labeled aptamer (biotin probe) and [Ru(bpy)3]2+ (TBR)-Au bio bar code-labeled aptamer (ECL nanoprobe), to form a double aptamer–protein sandwich complex. The complex is then immobilized on the streptavidin microbeads through biotin–streptavidin linkage and detected by ECL assay. The ECL signal of the target protein is amplified by the TBR-bio bar code DNAs. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 1pM of PDGF-BB. A calibration curve with a linearity range from 1pM to 10nM is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this aptamer-based bio bar code IM-ECL method holds great promise in protein detection.

Electrochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system

MicroRNAs (miRNAs), a kind of small, endogenous, noncoding RNAs (∼22 nucleotides), might play a crucial role in early cancer diagnose due to its abnormal expression in many solid tumors. As a result, label-free and PCR-amplification-free assay for miRNAs is of great significance. In this work, a highly sensitive biosensor for sequence specific miRNA-21 detection without miRNA-21 labeling and enrichment was constructed based on the substrate electrode of dendritic gold nanostructure (DenAu) and graphene nanosheets modified glassy carbon electrode. Sulfydryl functionalized locked nucleic acid (LNA) integrated hairpin molecule beacon (MB) probe was used as miRNA-21 capture probe. After hybridized with miRNA-21 and reported DNA loading in gold nanoparticles (AuNPs) and biotin multi-functionalized bio bar codes, streptavidin–HRP was brought to the electrode through the specific interaction with biotin to catalyze the chemical oxidation of hydroquinone by H2O2 to form benzoquinone. The electrochemical reduction signal of benzoquinone was utilized to monitor the miRNA-21 hybridization event. The effect of experimental variables on the amperometric response was investigated and optimized. Based on the specific confirmation of probe and signal amplification, the biosensor showed excellent selectivity and high sensitivity with low detection limit of 0.06pM. Successful attempts are made in miRNA-21 expression analysis of human hepatocarcinoma BEL-7402 cells and normal human hepatic L02 cells.

Nanogold-based bio-bar codes for label-free immunosensing of proteins coupling with an in situ DNA-based hybridization chain reaction

A label-free, non-enzyme immunosensing strategy is designed for ultrasensitive electronic detection of disease-related proteins (carcinoembryonic antigen as a model) by using gold nanoparticle-based bio-bar codes and an in situ amplified DNA-based hybridization chain reaction.

Nanotechnology for Alzheimer’s disease detection and treatment

In this paper, we present the role of nanotechnology in the development and improvement of techniques for early diagnosis and effective treatment of Alzheimer's disease (AD). Since AD pathology is almost irreversible and presentday medications for AD only lower its associated symptoms, application of diseasemodifying treatments could be successful only if AD early diagnosis is possible. The nanodiagnostic methods reported and compared in this paper include both of in vitro and in vivo nature. Of the in vitro approaches, the DNAnanoparticle conjugates (bioba rcode assay), nanoparticle surface plasmon resonance, scanning tunneling microscopy, and twophoton Rayleigh spectroscopy are presented here. Of the in vivo methods, µMRI and optical imaging techniques are discussed here. The nanotreatment methods for AD are numerous. They are categorized in this report under neuroprotective methods from toxicity of amyloid�β peptide (Aβ) oligomers , oxidative stress of free radicals and nanocarriers for targeted drug delivery. The important agents for neuroprotection include nanogels, fullerene, nanoceria, dendrimers, gold nanoparticles, and diamondoid derivatives. The major nanocarriers presented here include cholinesterase inhibitors nanocarriers, acetylcholine nanocarrier, metal chelator nanocarriers, (Iron chelators and copper chelators), curcuminoids nanocarrier, antioxidant nanocarriers, and gene nanocarriers. Considering that the AD is a multifactorial disease with several pathogenetic mechanisms and pathways, a multifunctional nanotechnology approach will be needed to target its main molecular culprits. These molecular targets must include, but not limited to, Aβ oligomers, reactive oxygen species (ROS), excessive metal ions, tau phosphorylating kinases and cell cycle proteins.

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequenc-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H2O2 and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L-1 of target DNA.

Construction of DNA sandwich electrochemical biosensor with nanoPbS and nanoAu tags on magnetic microbeads

A novel and sensitive sandwich electrochemical biosensor based on the amplification of magnetic microbeads and Au nanoparticles (NPs) modified with bio bar code and PbS nanoparticles was constructed in the present work. In this method, the magnetic microspheres were coated with 4 layers polyelectrolytes in order to increase carboxyl groups on the surface of the magnetic microbeads, which enhanced the amount of the capture DNA. The amino-functionalized capture DNA on the surface of magnetic microbeads hybridized with one end of target DNA, the other end of which was hybridized with signal DNA probe labelled with Au NPs on the terminus. The Au NPs were modified with bio bar code and the PbS NPs were used as a marker for identifying the target oligoncleotide. The modification of magnetic microbeads could immobilize more amino-group terminal capture DNA, and the bio bar code could increase the amount of Au NPs that combined with the target DNA. The detection of lead ions performed by anodic stripping voltammetry (ASV) technology further improved the sensitivity of the biosensor. As a result, the present DNA biosensor showed good selectivity and sensitivity by the combined amplification. Under the optimum conditions, the linear relationship with the concentration of the target DNA was ranging from 2.0 × 10 −14 M to 1.0 × 10 −12 M and a detection limit as low as 5.0 × 10 −15 M was obtained.

Electrochemical detection of DNA hybridization based on bio-bar code method

Based on the electrochemical detection of cadmium ions dissolved from CdS nanoparticles (NPs), a novel and sensitive assay for the sequence-specific DNA detection has been developed by using bio-bar code techniques. The “sandwich-type” DNA complexes were fabricated with the thiol-functionalized capture DNA firstly immobilized on the modified electrode (nanoAu-GCE) and hybridized with one end of target DNA, the other end of which was recognized with signal DNA labeled on the surface of Au NP. To amplify the detection signals, the Au NP was also modified with CdS NPs by an amidization reaction between bio-bar code binding DNA on the surface of Au NPs and mercaptoacetic acid on the surface of CdS NPs. The hybridization events were monitored by the electrochemical detection after the cadmium ions were dissolved from the hybrids. Under optimum conditions, the peak current value was increased with the increase of the concentration of target DNA in the range of 1.0 × 10 −14 to 1 .0 × 10 −13 M. A detection limit of 4.2 × 10 −15 M of target DNA was achieved. The ASV currents of two-base mismatched sequences and non-complementary sequences were also detected. The experiments indicated that two-base mismatched sequences showed weaker peak current and non-complementary sequences gave no response at all.