Development of a multilayered polymeric DNA biosensor using radio frequency technology with gold and magnetic nanoparticles

This study utilized the radio frequency (RF) technology to develop a multilayered polymeric DNA sensor with the help of gold and magnetic nanoparticles. The flexible polymeric materials, poly (p-xylylene) (Parylene) and polyethylene naphtholate (PEN), were used as substrates to replace the conventional rigid substrates such as glass and silicon wafers. The multilayered polymeric RF biosensor, including the two polymer layers and two copper transmission structure layers, was developed to reduce the total sensor size and further enhance the sensitivity of the biochip in the RF DNA detection. Thioglycolic acid (TGA) was used on the surface of the proposed biochip to form a thiolate-modified sensing surface for DNA hybridization. Gold nanoparticles (AuNPs) and magnetic nanoparticles (MNPs) were used to immobilize on the surface of the biosensor to enhance overall detection sensitivity. In addition to gold nanoparticles, the magnetic nanoparticles has been demonstrated the applicability for RF DNA detection. The performance of the proposed biosensor was evaluated by the shift of the center frequency of the RF biosensor because the electromagnetic characteristic of the biosensors can be altered by the immobilized multilayer nanoparticles on the biosensor. The experimental results show that the detection limit of the DNA concentration can reach as low as 10 pM, and the largest shift of the center frequency with triple-layer AuNPs and MNPs can approach 0.9 and 0.7 GHz, respectively. Such the achievement implies that the developed biosensor can offer an alternative inexpensive, disposable, and highly sensitive option for application in biomedicine diagnostic systems because the price and size of each biochip can be effectively reduced by using fully polymeric materials and multilayer-detecting structures.     

Gold nanoparticles modified electrode via simple electrografting of in situ generated mercaptophenyl diazonium cations for development of DNA electrochemical biosensor

A novel protocol for development of DNA electrochemical biosensor based on gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE) was proposed, which was carried out by the self-assembly of AuNPs on the mercaptophenyl film (MPF) via simple electrografting of in situ generated mercaptophenyl diazonium cations. The resulting MPF was covalently immobilized on GCE surface via C-C bond with high stability, which was desirable in fabrication of excellent performance biosensors. Probe DNA was self-assembled on AuNPs through the well-known Au-thiol binding. The recognition of fabricated DNA electrochemical biosensor toward complementary single-stranded DNA was determined by differential pulse voltammetry with the use of Co(phen)(3)(3+) as the electrochemical indicator. Taking advantage of amplification effects of AuNPs and stability of MPF, the developed biosensor could detect target DNA with the detection limit of 7.2×10(-11) M, which also exhibits good selectivity, stability and regeneration ability for DNA detection.     

Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry

A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.     

Enzyme-nanoparticle conjugates at oil-water interface for amplification of electrochemical immunosensing

A novel cholesterol biosensor was prepared based on gold nanoparticles-catalyzed luminol electrogenerated chemiluminescence (ECL). Firstly, l-cysteine-reduced graphene oxide composites were modified on the surface of a glassy carbon electrode. Then, gold nanoparticles (AuNPs) were self-assembled on it. Subsequently, cholesterol oxidase (ChOx) was adsorbed on the surface of AuNPs to construct a cholesterol biosensor. The stepwise fabrication processes were characterized with cyclic voltammetry and atomic force microscopy. The ECL behaviors of the biosensor were also investigated. It was found that AuNPs not only provided larger surface area for higher ChOx loading but also formed the nano-structured interface on the electrode surface to improve the analytical performance of the ECL biosensor for cholesterol. Besides, based on the efficient catalytic ability of AuNPs to luminol ECL, the response of the biosensor to cholesterol was linear range from 3.3 μM to 1.0 mM with a detection limit of 1.1 μM (S/N=3). In addition, the prepared ECL biosensor exhibited satisfying reproducibility, stability and selectivity. Taking into account the advantages of ECL, we confidently expect that ECL would have potential applications in biotechnology and clinical diagnosis.     

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH–ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA.     

Electrochemical detection of the MT-ND6 gene and its enzymatic digestion: Application in human genomic sample

A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome.     

Studies of the binding and signaling of surface-immobilized periplasmic glucose receptors on gold nanoparticles: a glucose biosensor application

Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur-gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV-Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 microM.     

Carbon nanotube/gold nanoparticles/polyethylenimine-functionalized ionic liquid thin film composites for glucose biosensing

A novel glucose biosensor based on immobilization of glucose oxidase (GOD) in thin films of polyethylenimine-functionalized ionic liquid (PFIL), containing a mixture of carbon nanotubes (CNT) and gold nanoparticles (AuNPs) and deposited on glassy carbon electrodes, was developed. Direct electrochemistry of glucose oxidase in the film was observed, with linear glucose response up to 12mM. The PFIL-stabilized gold nanoparticles had a diameter of 2.4+/-0.8nm and exhibited favorable stability (stored even over one month with invisible change in UV-vis spectroscopic measurements). In addition, CNT were also well dispersed in the PFIL matrix, then, the resulting CNT/AuNPs/PFIL composites film showed high electrocatalytic activity toward reduction of hydrogen peroxide and oxygen. Here, PFIL, due to its high ionic conductivity, good solubility to CNT, and stability to nanoparticles, played an important role in constructing stable CNT/AuNPs/PFIL/GOD composites. And good biocompatibility of PFIL also offered a friendly environment for the immobilization of biomolecules.     

A label-free electrochemiluminescence aptasensor for thrombin based on novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles

In the work, a label-free electrochemiluminescence (ECL) aptasensor for the sensitive and selective detection of thrombin was constructed based on target-induced direct ECL signal change by virtue of a novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles (luminol-AuNPs). It is the first label-free ECL biosensor based on luminol and its analogs functionalized AuNPs. Streptavidin AuNPs coated with biotinylated DNA capture probe 1 (AuNPs-probe 1) were firstly assembled onto an gold electrode through 1,3-propanedithiol. Then luminol-AuNPs co-loaded with thiolated DNA capture probe 2 and thiolated thrombin binding aptamer (TBA) (luminol-AuNPs-probe 2/TBA) were assembled onto AuNPs-probe 1 modified electrode through the hybridization between capture probes 1 and 2. The luminol-AuNPs-probe 2/TBA acted as both molecule recognition probe and sensing interface. An Au/AuNPs/ds-DNA/luminol-AuNPs/TBA multilayer architecture was obtained. In the presence of target thrombin, TBA on the luminol-AuNPs could capture the thrombin onto the electrode surface, which produced a barrier for electro-transfer and influenced the electro-oxidation reaction of luminol, leading to a decrease in ECL intensity. The change of ECL intensity indirectly reflected the concentration of thrombin. Thus, the approach showed a high sensitivity and a wider linearity for the detection of thrombin in the range of 0.005-50nM with a detection limit of 1.7pM. This work reveals that luminol-AuNPs are ideal platform for label-free ECL bioassays.     

Cationic Surfactant-Based Colorimetric Detection of Plasmodium Lactate Dehydrogenase,a Biomarker for Malaria,Using the Specific DNA Aptamer

A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum.     

Label-free detection of bacterial RNA using polydiacetylene-based biochip

A novel acetylcholinesterase (AChE)/choline oxidase (ChOx) bienzyme amperometric acetylcholine biosensor based on gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWCNTs) has been successfully developed by self-assembly process in combination of sol-gel technique. A thiolated aqueous silica sol containing MWCNTs and ChOx was first dropped on the surface of a cleaned Pt electrode, and then AuNPs were assembled with the thiolated sol-gel network. Finally, the alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and AChE was repeated to assemble different layers of PDDA-AChE on the electrode for optimizing AChE loading. Among the resulting biosensors, the biosensor based on two layers of PDDA-AChE multilayer films showed the best performance. It exhibited a wide linear range, high sensitivity and fast amperometric response, which were 0.005-0.4mM, 3.395 μA/mM, and within 15s, respectively. The biosensor showed long-term stability and acceptable reproducibility. More importantly, this study could provide a simple and effective multienzyme immobilization platform for meeting the demand of the effective immobilization enzyme on the electrode surface.     

Ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based DNA amplification

The present study describes an ultrasensitive protein biochip that employs nanogap electrodes and self-assembled nanoparticles to electrically detect protein. A bio-barcode DNA technique amplifies the concentration of target antigen at least 100-fold. This technique requires the establishment of conjugate magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) through binding between monoclonal antibodies (2B2), the target antigen, and polyclonal antibodies (GP). Both GP and capture ssDNA (single-strand DNA) bonds to bio-barcode ssDNA are immobilized on the surface of AuNPs. A denature process releases the bio-barcode ssDNAs into the solution, and a hybridization process establishes multilayer AuNPs over the gap surface between electrodes. Electric current through double-layer self-assembled AuNPs is much greater than that through self-assembled monolayer AuNPs. This significant increase in electric current provides evidence that the solution contains the target antigen. Results show that the protein biochip attains a sensitivity of up to 1 pg/μL.     

Size-dependent plasmonic responses of single gold nanoparticles for analysis of biorecognition

We report the use of plasmonic responses of single gold nanoparticles (AuNPs) with various sizes for the analysis of biomolecular recognition. We also describe the relationship between particle size and plasmonic response induced by the binding of receptors and target analytes. To investigate the plasmonic response of AuNPs, Rayleigh light scattering spectra were collected from individual AuNPs using a dark-field microspectroscopy system. Using prostate-specific antigen (PSA) as a model, the linear dynamic range was obtained in the concentration range of 10(-4) to 10 ng/ml, with the smallest detectable concentration at 0.1 pg/ml corresponding to localized surface plasmon resonance (LSPR) λ(max) shifts of approximately 2.95 nm. This result indicates that individual AuNPs can be used for development of a very sensitive, robust, simple, and label-free biosensor to detect protein biomarkers. Furthermore, the method possesses great potential for monitoring other biological interactions.     

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: a femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG3-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and FT-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG6-COOH and HS-OEG3-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.     

Visible-light Induced Reduction of Graphene Oxide Using Plasmonic Nanoparticle

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using visible light irradiation with plasmonic nanoparticles. The plasmonic nanoparticle is used to improve the reduction efficiency of GO. It only takes 30 min at RT by illuminating the solutions with Xe-lamp, the r-GO solutions can be obtained by completely removing gold nanoparticles through simple centrifugation step. The spherical gold nanoparticles (AuNPs) as compared to the other nanostructures is the most suitable plasmonic nanostructure for r-GO preparation. The reduced graphene oxide prepared using visible light and AuNPs was equally qualitative as chemically reduced graphene oxide, which was supported by various analytical techniques such as UV-Vis spectroscopy, Raman spectroscopy, powder XRD and XPS. The reduced graphene oxide prepared with visible light shows excellent quenching properties over the fluorescent molecules modified on ssDNA and excellent fluorescence recovery for target DNA detection. The r-GO prepared by recycled AuNPs is found to be of same quality with that of chemically reduced r-GO. The use of visible light with plasmonic nanoparticle demonstrates the good alternative method for r-GO synthesis.     

Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy

An electrochemical biosensor based on functional composite nanofibers for hybridization detection of specific K-ras gene that is highly associated with colorectal cancer via multiple signal amplification strategy has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped nylon 6 (PA6) composite nanofibers (MWCNTs–PA6) was prepared using electrospinning, which served as the nanosized backbone for thionine (TH) electropolymerization. The functional composite nanofibers [MWCNTs–PA6–PTH, where PTH is poly(thionine)] used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. Through the hybridization reaction, a sandwich format of ssDNA1/K-ras gene/gold nanoparticle-labeled ssDNA2 (AuNPs–ssDNA2) was fabricated, and the AuNPs offered excellent electrochemical signal transduction. The signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles (TA/AuNPs). A significant sensitivity enhancement was obtained; the detection limit was down to 30 fM, and the discriminations were up to 54.3 and 51.9% between the K-ras gene and the one-base mismatched sequences including G/C and A/T mismatched bases, respectively. The amenability of this method to the analyses of K-ras gene from the SW480 colorectal cancer cell lysates was demonstrated. The results are basically consistent with those of the K-ras Kit (HRM: high-resolution melt). The method holds promise for the diagnosis and management of cancer.     

Surface Plasmon Resonance Studies of the Hybridization Behavior of DNA-Modified Gold Nanoparticles with Surface-Attached DNA Probes

Colloidal gold nanoparticles (AuNPs) have been extensively investigated as amplification tags to improve the sensitivity of surface plasmon resonance (SPR) biosensors. When using the so-called AuNP-enhanced SPR technique for DNA detection, the density of single-stranded DNA (ssDNA) on both the AuNPs and planar gold substrates is of crucial importance. Thus, in this work, we carried out a systematical study about the influence of surface ssDNA density onto the hybridization behavior of various DNA-modified AuNPs (DNA-AuNPs) with surface-attached DNA probes by using surface plasmon resonance spectroscopy. The lateral densities of the ssDNA on both the AuNPs and planar gold substrates were controlled by using different lengths of oligo-adenine sequence (OAS) as anchoring group. Besides SPR measurements, the amount of the captured DNA-AuNPs after the hybridization was further identified via atomic force microscope (AFM). SPR and AFM results clearly indicated that a higher ssDNA density on either the AuNPs or the gold substrates would give rise to better hybridization efficiency. Moreover, SPR data showed that the captured DNA-AuNPs could not be removed from SPR sensor surfaces using various dehybridization solutions regardless of surface ssDNA density. Consequently, it is apparent that the hybridization behavior of DNA-AuNPs was different from that of solution-phase ssDNA. Based on these data, we hypothesized that both multiple recognitions and limited accessibility might account for the hybridization of DNA-AuNPs with surface-attached ssDNA probes.

     

Size- and coating-dependent uptake of polymer-coated gold nanoparticles in primary human dermal microvascular endothelial cells

A library-orientated approach is used to gain understanding of the interactions of well-defined nanoparticles with primary human endothelial cells, which are a key component of the vasculature. Fifteen sequentially modified gold nanoparticles (AuNPs) based on three different core sizes (18, 35, 65 nm) and five polymeric coatings were prepared. The synthetic methodology ensured homogeneity across each series of particles to allow sequential investigation of the chemical features on cellular interactions. The toxicity of these nanoparticles, their uptake behavior in primary human dermal microvascular endothelial cells (HDMECs), and quantification of uptake were all investigated. The results of our studies indicated that high concentrations of gold nanoparticles (250 μg/mL) were nontoxic and that the number of internalized nanoparticles was related to nanoparticle size and surface chemistry. In summary, the positive-charged ethanediamine-coated AuNPs were internalized to a greater extent than the negative- or neutral-charged AuNPs. Moreover, differences in the amounts of internalized AuNPs could be shown for the three neutral-charged AuNPs, whereas the uptake of hydroxypropylamine-coated particles was preferred compared with glucosamine-coated or PEGylated AuNPs. Hydroxypropylamine-coated AuNPs were found to be the most efficient neutral-charged particles in overcoming the endothelial cell barrier and entering the cell.     

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 H(2)O(2) 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 0.06 pM. Successful attempts are made in miRNA-21 expression analysis of human hepatocarcinoma BEL-7402 cells and normal human hepatic L02 cells.     

Selective detection of silver ions using mushroom-like polyaniline and gold nanoparticle nanocomposite-based electrochemical DNA sensor

A highly sensitive electrochemical DNA biosensor made of polyaniline (PANI) and gold nanoparticles (AuNPs) nanocomposite (AuNPs@PANI) has been used for the detection of trace concentration of Ag⁺. In the presence of Ag⁺, with the interaction of cytosine–Ag⁺–cytosine (C–Ag⁺–C), cytosine-rich DNA sequence immobilized onto the surface of AuNPs@PANI has a self-hybridization and then forms a duplex-like structure. The whole detection procedure of Ag⁺ based on the developed biosensor was evaluated by electrochemical impedance spectroscopy. On semi-logarithmic plots of the log Ag⁺ concentration versus peak current, the results show that the prepared biosensor can detect silver ions at a wide linear range of 0.01–100 nM (R = 0.9828) with a detection limit of 10 pM (signal/noise = 3). Moreover, the fabricated sensor exhibits good selectivity and repeatability. The detection of Ag⁺ was determined by Ag⁺ self-induced conformational change of DNA scaffold that involved only one oligonucleotide, showing its convenience and availability.