Streptavidin-modified Gold Nanoparticles Research Articles

The Contribution of Polyaniline and Gold Nanoparticles for Enhancing Current Signals of Streptavidin Based Immunosensors

The presence of streptavidin has been found to serve as an electrically insulating layer. This could reduce the initial current response of an electrochemical immunosensor, reducing its performancefor detecting the analyte of interest. The work aimed to investigate the current response of the streptavidin based electrochemical immunosensor at polyaniline and gold nanoparticle modified screen-printed electrodes and compare to without gold nanoparticle addition. The result of the streptavidin modified gold nanoparticles - polyaniline sensor exhibited a three-times higher current response than that of the streptavidin modified polyaniline sensor (without gold nanoparticle).Additionally, antibody immobilization (EpCAM as a model) on the modified electrode showed high performances for use further in antigen target detection. The high conductivity of the developed immunosensor could become a promising candidate for immunosensor development with high sensitivity in the future.

Surface plasmon resonance biosensor for the detection of miRNAs by combining the advantages of homogeneous reaction and heterogeneous detection

The hybridization and enzymolysis reactions for nucleic acid detection were carried out on the chip surface in the traditional surface plasmon resonance (SPR) biosensors. Herein, we proposed an innovative method for microRNA (miRNA) detection in which the hybridization-enzymolysis recycling reactions were performed in solution. Duplex-specific nuclease (DSN) and streptavidin-modified gold nanoparticles (SA-AuNPs) were employed for enhancing the assay sensitivity. In the absence of miRNA, the biotinylated DNA probe (bio-DNA-bio, biotin tags at both the 3′ and 5′ termini of DNA) was attached to the SA-modified chip through the SA-biotin binding, allowing the capture of SA-AuNPs with the same interaction. As a result, a larger SPR signal was attained. However, in the presence of miRNA, bio-DNA-bio hybridized with miRNA was digested by DSN. In this process, the miRNA strand remained intact and participated in the next hybridization-enzymolysis recycling process. Thus, one miRNA could promote the hydrolysis of many bio-DNA-bio probes and allow the generation of numerous bio-DNA fragments. Meanwhile, the produced bio-DNA competed with the undigested bio-DNA-bio to bind SA on the chip surface. The digestion of bio-DNA-bio and the competitive binding between bio-DNA-bio and bio-DNA led to the attachment of fewer SA-AuNPs and then smaller SPR signals. The change in SPR signal at the concentration as low as 1 fM miRNA has been readily determined. The strategy possessed the advantageous properties of simple operation, fast response, high sensitivity and excellent specificity, serving as a viable means for the fabrication of novel sensing platforms.

Colorimetric nanoplatform for visual determination of cancer cells via target-catalyzed hairpin assembly actuated aggregation of gold nanoparticles.

According to aptamer-mediated hairpin DNA cascade amplifier and gold nanoparticles aggregation, an optical platform for cancer cells determination has been proposed. High-affinity chimeric aptamers were used for cancer cell detection and also as an initiator for beginning hairpin assembly to construct three-way junction (3WJ) nanostructures. These three hairpins were modified at 3' ends with biotin. In the presence of target cell, chimeric aptamer binds to its ligand on cell surface and initiates 3WJ nanostructures formation. These 3WJ nanostructures interact with streptavidin-modified gold nanoparticles (AuNPs) via non-covalent biotin-streptavidin interactions and create a crossover lattice of nanoparticles. This event leads to AuNPs aggregation and red-shifting. The results were confirmed by gel electrophoresis and UV-visible spectrophotometry. The dynamic range of this assay is 25 to 107 cells with a detection limit of 10 cells which is respectively 9 and 4 times more significant than the sensitivity of AuNP-based approaches without amplification and enzyme-mediated signal amplification. Graphical abstract.

Biotinylated Phosphorus Dendrimers as Control Line in Nucleic Acid Lateral Flow Tests

Lateral flow assays (LFA) are an affordable, easy-to-use, qualitative rapid test for clinical diagnosis in nonlaboratory environments and low-resource facilities. The control line of these tests is very important to provide a valid result, confirming that the platform operates correctly. A clear, nondiffused line is desirable. The number of colored nanoparticles that reach the control line in a positive test can be very small, and they should all be trapped efficiently by the molecules adsorbed there. In this work, we proposed the use of robust biotinylated dendrimers of two different generations as signal amplifiers in control lines of LFA, able to react with streptavidin-modified gold nanoparticles. Besides the synthesis and characterization, the analytical performance as control lines will be studied, and their response will be compared with other commercially available biotinylated molecules. Finally, the utility of the dendrimer implemented in a NALF (Nucleic Acid Lateral Flow) strip was also demonstrated for detection of the amplicons obtained by double-tagging PCR (polymerase chain reaction) for the detection of E. coli as a model of foodborne pathogen.

A highly sensitive and specific lateral flow aptasensor for the detection of human osteopontin

The rapid determination of human osteopontin (OPN) protein, a potential cancer biomarker, holds substantial promise for point-of-care diagnostics and biomedical applications. To date, most reported platforms for OPN detection are apparatus-dependent, time-consuming, and expensive. Herein, we established a lateral flow biosensor (LFB) for OPN detection. A biotinylated aptamer was used for OPN pre-capture from samples, an antibody for OPN was immobilized on the test line for a second specific target identification, and streptavidin-modified gold nanoparticles were sprayed on the conjugation pad for color detection. This LFB achieved as low as 0.1 ng mL−1 OPN sensitivity with a good dynamic detection between 10 and 500 ng mL−1 within 5 min. Intriguingly, the LFB allowed a qualitative and semi-quantitative detection of OPN in serum at clinically cut-off levels as in cancer patients, and can discriminate OPN from interfering proteins with high specificity. Thus, it is a promising alterative approach for point-of-care OPN screening and detection.

Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles.

Loop-mediated isothermal amplification (LAMP) eradicates the need of thermocycler in DNA amplification. Signals are usually obtained via fluorometry or turbidimetry, but such methods need improvement in order to become more effortless and reliable. The authors describe a set of six specific primers targeting the species-specific tlh gene of Vibrio parahaemolyticus which were used in accelerated LAMP reaction. Gold nanoparticles (AuNPs) were functionalized with streptavidin (Avidin-AuNPs), and engineered to signal the LAMP reaction. Two of the loop primers for LAMP were biotinylated and then can produce a DNA that can cause clusterization of Avidin-AuNPs based on the formation of avidin-biotin complex. This leads to a color change of the solution from red to blue. Amplification is completed within 30min and can be visually detected within 5min. The detection limit of the method is found to be 8.6cfu per reaction. This visual detection scheme does not require any fluorescent reagents and detection instruments. Conceivably, the method has a wide scope because such Avidin-AuNPs can be used as nanoprobes for a variety of other LAMP products. This rapid and universal strategy holds promise in point of care testing and food testing, particularly in resource-limited regions. Graphical abstract Six specific primers (two of them are biotinylated) were used to realize the accelerated Loop-Mediated Isothermal Amplification. Streptavidin modified gold nanoparticles (Avidin-AuNPs) cluster on the DNA products, leading to the apparent change of color from red to blue, which is readily identified even by unaided eye.

Sensitive detection of carcinoembryonic antigen using surface plasmon resonance biosensor with gold nanoparticles signal amplification

A new method for real-time detection of carcinoembryonic antigen (CEA) in human serum with high sensitivity and selectivity using surface plasmon resonance (SPR) biosensor was developed. Two kinds of antibodies were used to recognize CEA at different epitopes with high affinity and specificity. Gold nanoparticles (GNPs) modified with streptavidin (SA) were used to further enhance signal specifically via biotin–streptavidin interaction. The binding capacity of the streptavidin-modified gold nanoparticles (SA–GNPs) for ligand biotin was quantified by titration with biotin (5-fluorescein) conjugate to be 10.54 biotin binding sites per 100nm2. The developed GNPs enhanced sandwich SPR biosensor successfully fulfilled the sensitive detection of CEA in the range of 1–60ng/mL with a detection limit of 1.0ng/mL. Compared to the direct assay format, sandwich format without GNPs and SA–GNPs enhanced sandwich format led to 4.2-fold and 13.8-fold in the sensitivity, respectively. This sensor also showed good selectivity for CEA in the interference study. The results demonstrated that the proposed method could provide a high sensitivity and selectivity in the detection of CEA and offer a promising alternative for cancer biomarker than traditional clinical examinations.

The detection of a transgenic soybean biochip using gold label silver stain technology

A method for the rapid detection of transgenic soybean crops based on a combination of gene chip and "gold label silver stain" (GLSS) technologies has been established. To ensure the specificity of this method, the CaMV35S promoter and Nos terminator were selected as probes because they are both exogenous genes that are specific to transgenic soybean plants. The addition of biotin-modified dUTPs to a polymerase chain reaction (PCR) system can produce amplified nucleic acid segments containing biotin. These labeled PCR products then hybridize with specific probes on the chip and are subsequently bound by streptavidin-modified gold nanoparticles (GNPs). Due to the catalytic nature of the GNPs, silver staining can be used to visualize the hybridized probes, which appear as signals in varying shades of gray. The intensity value of the gray signals can be obtained using a general scanner. Silver staining for 10 min was determined to produce the optimal signal-to-noise ratio. In addition, this method was shown to be highly specific and had a detection sensitivity of 288.57 pg/μL.

DNA sensor by using electrochemiluminescence of acridinium ester initiated by tripropylamine

It was found that tripropylamine (TPA) could be used as a coreactant to initiate the electrochemiluminescence (ECL) of acridinium NHS ester (AE NHS) labels attached to DNA. The radicals generated in the electro-oxidation process of TPA reacted with AE NHS to form the excited N-methylacridone, giving rise to light emission. The AE/TPA ECL system was for the first time used as the detection system for developing an ECL-based DNA sensor. In the protocol, streptavidin-modified gold nanoparticles were firstly immobilized onto a thiol-treated gold electrode. The streptavidin could specifically interact with the biontinylated capture DNA. Afterwards, the target DNA and the AE-labeled report DNA were conjugated onto the electrode step by step due to the hybridization reactions, and a sandwich-type sensor was fabricated. The ECL signals of the sensor were obtained under pulse potential condition in alkaline solution containing 50.0 mmol L(-1) TPA. Under optimized experimental conditions, the linear range of the DNA sensor for the determination of the target DNA was from 5.0 × 10(-15) to 5.0 × 10(-12) mol L(-1). The detection limit (S/N = 3) was 3.0 × 10(-15) mol L(-1). Moreover, the sensor could specifically recognize the target DNA against one base-pair mismatched sequences, two base-pair mismatched sequences, and the noncomplementary sequences. It is of great application potential in clinic analysis.

Impedimetric genosensing of DNA polymorphism correlated to cystic fibrosis: A comparison among different protocols and electrode surfaces

In this work, a genosensor for the impedimetric detection of the triple base deletion in a cystic fibrosis-related DNA synthetic sequence is presented. Screen-Printed Carbon Electrodes containing Carboxyl functionalised multi-walled carbon nanotubes were used for the immobilization of an amino-modified oligonucleotide probe, complementary to the Cystic Fibrosis (CF) mutant gene. The complementary target (the mutant sequence) was then added and its hybridization allowed. The change of interfacial charge transfer resistance ( R ct) between the solution and the electrode surface, experimented by the redox marker ferrocyanide/ferricyanide, confirmed the hybrid formation. A non-complementary DNA sequence and a three-mismatch sequence corresponding to the wild DNA gene (present in healthy people) were used as negative controls. A further step employing a signalling biotinylated probe was performed for signal amplification using streptavidin-modified gold nanoparticles (strept-AuNPs). In order to observe by SEM the presence and distribution of strept-AuNPs, a silver enhancement treatment was applied to electrodes already modified with DNA–nanoparticles conjugate. The developed protocol allowed the very sensitive detection of the triple base deletion in a label-free CF-related DNA sequence, achieving a LOD around 100 pM. Results were finally compared with those obtained using different protocols for immobilization of DNA capture probe.

DNA hybridization detection by electrochemical impedance spectroscopy using interdigitated gold nanoelectrodes

A DNA biosensor is presented that is based on gold interdigitated nanoelectrodes of 100 nm width and 250 nm pitch. A single-strand oligonucleotide acts as the capture probe. Because of its nanometer dimensions, the device shows improved sensitivity when compared to similar systems. This encouraged us to perform a direct and unlabelled detection. After incubation with the DNA target, the impedance spectrum was recorded between 1 kHz and 10 MHz to obtain the net capacitance change. The use of a biotinylated DNA signalling probe permitted the integration of an amplification stage in a sandwich format that employs streptavidin-modified gold nanoparticles. The strategy was preliminarily tested by detecting the breast cancer related BRCA1 gene, where the noncomplementary, wild and mutated forms were easily differentiated at a concentration level of 3 μM (corresponding to a 30 pmol quantity).

Impedimetric genosensors employing COOH-modified carbon nanotube screen-printed electrodes

Screen-printed electrodes modified with carboxyl functionalised multi-walled carbon nanotubes were used as platforms for impedimetric genosensing of oligonucleotide sequences specific for transgenic insect resistant Bt maize. After covalent immobilization of aminated DNA probe using carbodiimide chemistry, the impedance measurement was performed in a solution containing the redox marker ferrocyanide/ferricyanide. A complementary oligomer (target) was then added, its hybridization was promoted and the measurement performed as before. The change of interfacial charge transfer resistance between the solution and the electrode surface, experimented by the redox marker at the applied potential, was recorded to confirm the hybrid formation. Non-complementary DNA sequences containing a different number of base mismatches were also employed in the experiments in order to test specificity. A signal amplification protocol was then performed, using a biotinylated complementary target to capture streptavidin modified gold nanoparticles, thus increasing the final impedimetric signal (LOD improved from 72 to 22 fmol, maintaining a good reproducibility, in fact RSD < 12.8% in all examined cases). In order to visualize the presence and distribution of gold nanoparticles, a silver enhancement treatment was applied to electrodes already modified with DNA–nanoparticles conjugate, allowing direct observation by scanning electron microscopy.

Design and Fabrication of Nanostructures Based on DNA Ring–Protein Complex

In this report, we describe the design and fabrication of DNA nanostructures (“DNA glasses”, “DNA serial rings”, and “DNA chains”) using DNA ring–protein complexes. An experiment was performed to fabricate DNA ring-conjugated biotin using two types of DNA (“vector DNA” and “insert DNA”). The vector DNA was obtained by cutting plasmid DNA with restriction enzymes. The insert DNA (DNA conjugated with biotin) was obtained using a DNA synthesizer. After ligation and the introduction of streptavidin-modified gold nanoparticles, DNA structures were obtained. Subsequently, the DNA structures were observed by atomic force microscopy (AFM).

Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays

Prostate-specific antigen (PSA) is a valuable biomarker for prostate cancer screening. We developed a PSA immunoassay on a commercially available surface plasmon resonance biosensor. Our PSA receptor molecule consists of a single domain antigen-binding fragment, cAbPSA-N7, derived from dromedary heavy-chain antibodies and identified after phage display. It binds PSA with a high k(on) value of 1.9x10(6) M-1 s-1, and was covalently immobilised on a gold substrate via a mixed self-assembled monolayer (SAM) of alkanethiols by using carbodiimide-coupling chemistry in 10mM acetate buffer pH 5.5 to obtain an optimal pre-concentration. The best performing and optimised mixed SAM consisted of (10%) 16-mercapto-1-hexadecanoic acid (16-MHA) for covalent cAbPSA-N7 immobilisation and (90%) 11-mercapto-1-undecanol (11-MUOH) to minimise non-specific adsorption of the analyte. In this way, two advantages are incorporated in a single coupling layer. Up to 28 fmol/mm2 of cAbPSA-N7 could be immobilised and 30% of its binding sites participate actively in PSA interaction. In addition, the optimised layer showed also optimal performance to assess physiological samples. Although PSA concentrations as low as 10 ng/ml could be detected directly, this detection limit could be enhanced to PSA levels in the sub ng/ml range by introducing a sandwich assay involving a biotinylated secondary antibody and streptavidin modified gold nanoparticles. This approach realizes the PSA detection at clinical relevant concentrations.