Ultrasensitive and selective colorimetric detection of uric acid using peroxidase mimetic activity of biogenic palladium nanoparticles

Uric acid (2,6,8-trihydroxypurine) is a metabolic product of purine, which is one of the important markers of human health. The development of a rapid, facile, highly sensitive, and selective method for uric acid detection is critical for the diagnosis of related diseases and is still a strategic challenge. In this study, we developed a highly sensitive and selective colorimetric assay for the detection of uric acid using biogenic palladium nanoparticles (Pd NPs). The synthesized nanoparticles were shown to acquire peroxidase mimetic activity that oxidized 3,3′,5,5′-tetramethylbenzidine and produced a blue colour in an assay. The developed colorimetric assay is instrument-free detection of uric acid with a limit of detection of 0.05 μM and a 1.11 μM limit of quantification (LOQ). This is the first report determining the LOQ for a colorimetric assay that gives the lowest quantity of analyte that can be evaluated with more precision under the specified conditions of the analysis. The developed assay had a linear response at low uric acid concentrations of 0.05 to 1 μM and a 0.99841 linear regression correlation coefficient. This colorimetric detection provides a rapid, cost-effective, and easy-to-use platform for the clinical diagnosis of uric acid biomarkers.     

Ultrasensitive and selective detection of copper (II) and mercury (II) ions by dye-coded silver nanoparticle-based SERS probes

A simple and distinctive method for the ultrasensitive detection of Cu(2+) and Hg(2+) based on surface-enhanced Raman scattering (SERS) using cysteine-functionalized silver nanoparticles (AgNPs) attached with Raman-labeling molecules was developed. The glycine residue in a silver nanoparticle-bound cysteine can selectively bind with Cu(2+) and Hg(2+) and form a stable inner complex. Silver nanoparticles co-functionalized with cysteine and 3,5-Dimethoxy-4-(6'-azobenzotriazolyl)phenol (AgNP conjugates) can be used to detect Cu(2+) and Hg(2+) based on aggregation-induced SERS of the Raman tags. The addition of SCN(-) to the analyte can successfully mask Hg(2+) and allow for the selective detection of Cu(2+). This SERS-based assay showed an unprecedented limit of detection (LOD) of 10pM for Cu(2+) and 1pM for Hg(2+); these LODs are a few orders of magnitude more sensitive than the typical colorimetric approach based on the aggregation of noble nanoparticles. The analysis of real water samples diluted with pure water was performed and verified this conclusion. We envisage that this SERS-based assay may provide a general and simple approach for the detection of other metal ions of interest, which can be adopted from their corresponding colorimetric assays that have already been developed with significantly improved sensitivity and thus have wide-range applications in many areas.     

Ultrasensitive detection of rare mutations using next-generation targeted resequencing

With next-generation DNA sequencing technologies, one can interrogate a specific genomic region of interest at very high depth of coverage and identify less prevalent, rare mutations in heterogeneous clinical samples. However, the mutation detection levels are limited by the error rate of the sequencing technology as well as by the availability of variant-calling algorithms with high statistical power and low false positive rates. We demonstrate that we can robustly detect mutations at 0.1% fractional representation. This represents accurate detection of one mutant per every 1000 wild-type alleles. To achieve this sensitive level of mutation detection, we integrate a high accuracy indexing strategy and reference replication for estimating sequencing error variance. We employ a statistical model to estimate the error rate at each position of the reference and to quantify the fraction of variant base in the sample. Our method is highly specific (99%) and sensitive (100%) when applied to a known 0.1% sample fraction admixture of two synthetic DNA samples to validate our method. As a clinical application of this method, we analyzed nine clinical samples of H1N1 influenza A and detected an oseltamivir (antiviral therapy) resistance mutation in the H1N1 neuraminidase gene at a sample fraction of 0.18%.     

Ultrasensitive detection of proteins and antibodies by absorption-based laser wave-mixing detection using a chromophore label

Nonlinear laser wave mixing is presented as an ultrasensitive absorption-based method for the detection of proteins and antibodies using a nonfluorescing chromophore label, Coomassie Brilliant Blue (CBB). The complexes are flowed through a 150-μm (i.d.) capillary cell and detected using a low-power He-Ne laser. The wave-mixing signal is detected after 10 min of room temperature incubation for the antibody complex and after 18 min for the protein complex. All solutions are prepared in an aqueous buffer without the addition of organic modifiers. Concentration detection limits of 3.4 × 10^-19 and 6.4 × 10^-14 M (signal-to-noise ratio [S/N] = 2) are determined for bovine serum albumin (BSA) and human papillomavirus (HPV) antibody, respectively. Based on the small laser probe volume used (i.e., overlap volume of the two input beams), mass detection limits of 1.7 × 10^-22 and 2.6 × 10^-17 mol are determined for BSA and HPV antibody, respectively.     

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.     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using nanoCuS tags

A novel and sensitive biosensor for the determination of short sequence of DNA based on flow injection (FI)-chemiluminescence (CL) system of luminol-H2O2-Cu2+ was developed in the present work. The DNA probe labeled with copper sulfide nanoparticles (CuS NPs) could hybridize with target DNA immobilized on glass-carbon electrode (GCE). The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu2+ after the cupric ions was dissolved from the hybrids. A preconcentration process of cupric ions was performed by anodic stripping voltammetry (ASV) technology to improve the sensitivity of the biosensor. Under the optimum conditions, the CL intensity was proportional to the concentration of target DNA in the range of 2.0 x 10(-12)-1.0 x 10(-10)M. A detection limit of 5.5 x 10(-13)M of target DNA was achieved. The CL intensity of two-base mismatched sequences and noncomplementary sequences were also detected. The experiments indicated that two-base mismatched sequences showed weaker CL intensity and noncomplementary sequences gave no response at all.     

Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire

Cortisol is a member of the glucocorticoid hormone family and a key metabolic regulator. Increased intracellular cortisol levels have been implicated in type 2 diabetes, obesity, and metabolic syndrome. Cortisol is an important bio-marker of stress and its detection is also important in sports medicine. However, rapid methods for sensitive detection of cortisol are limited. Functionalized gold nanowires were used to enhance the sensitivity and selectivity of cortisol detection. Gold nanowires are used to improve the electron transfer between the electrodes. Moreover, the large surface to volume ratio, small diffusion time and high electrical conductivity and their aligned nature will enhance the sensitivity and detection limit of the biosensor several fold. The biosensor was fabricated using, aligned gold (Au) nanowires to behave as the working electrode, platinum deposited on a silicon chip to function as the counter electrode, and silver/silver chloride as reference electrode. The gold nanowires were coupled with cortisol antibodies using covalent linkage chemistry and a fixed amount of 3alpha-hydroxysteroid dehydrogenase was introduced into the reaction cell during each measurement to convert (reduce) ketosteroid into hydroxyl steroid. Furthermore, the micro-fluidic, micro-fluid part of the sensor was fabricated using micro-electro-mechanical system (MEMS) technology to have better control on liquid flow over Au nanowires to minimize the signal to noise ratio. The biosensor was characterized using SEM, AFM and FTIR technique. The response curve of the biosensor was found to be linear in the range of 10-80 microM of cortisol. Moreover, the presence of hydrocortisone is sensitively detected in the range of 5-30 microM. It is concluded that the functionalized gold nanowires with micro-fluidic device using enzyme fragment complementation technology can provide an easy and sensitive assay for cortisol detection in serum and other biological fluids.     

Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor

A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 μm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12) M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.     

Ultrasensitive staining-free protein detection after PAA gel electrophoresis using deep UV fluorescence

We present the observation of separated protein bands after polyacrylamide (PAA) gel electrophoresis based on the staining-free detection of their ultra violet (UV)-induced fluorescence employing deep UV confocal fluorescence microscopy. Mixtures of the three biological compounds beta-Galactosidase (from Escherichia coli), apo-Transferrin (bovine) and bovine serum albumin (BSA) have been separated and a staining free detection limit below 80 pg (7.0 x 10(8) molecules) per band has been achieved. This corresponds to approximately 270 molecules in the detection volume for confocal microscopy.     

A novel non-enzymatic ECL sensor for glucose using palladium nanoparticles supported on functional carbon nanotubes

A novel non-enzymatic electrochemiluminescence (ECL) sensor based on palladium nanoparticles (PdNPs)–functional carbon nanotubes (FCNTs) was discovered for glucose detection. PdNPs were homogeneously modified on FCNTs using a facile spontaneous redox reaction method. Their morphologies were characterized by transmission electron microscopy (TEM). Based on ECL experimental results, the PdNPs–FCNTs–Nafion film modified electrode displayed high electrocatalytic activity towards the oxidation of glucose. The free radicals generated by the glucose oxidation reacted with the luminol anion (LH⁻), and enhanced the ECL signal. Under the optimized conditions, the linear response of ECL intensity to glucose concentration was valid in the range from 0.5 to 40 μmol L⁻¹ (r² = 0.9974) with a detection limit (S/N = 3) of 0.09 μmol L⁻¹. In addition, the modified electrode presented high resistance towards the poisoning of chloride ion, high selectivity and long-term stability. In order to verify the sensor reliability, it was applied to the determination of glucose in glucose injection samples. The results indicated that the proposed approach provided a highly sensitive, more facile method with good reproducibility for glucose determination, promising the development of a non-enzymatic ECL glucose sensor.     

Ultrasensitive and label-free detection of pathogenic avian influenza DNA by using CMOS impedimetric sensors

This work presents miniaturized CMOS (complementary metal oxide semiconductor) sensors for non-faradic impedimetric detection of AIV (avian influenza virus) oligonucleotides. The signal-to-noise ratio is significantly improved by monolithic sensor integration to reduce the effect of parasitic capacitances. The use of sub-μm interdigitated microelectrodes is also beneficial for promoting the signal coupling efficiency. Capacitance changes associated with surface modification, functionalization, and DNA hybridization were extracted from the measured frequency responses based on an equivalent-circuit model. Hybridization of the AIV H5 capture and target DNA probes produced a capacitance reduction of -13.2 ± 2.1% for target DNA concentrations from 1 fM to 10 fM, while a capacitance increase was observed when H5 target DNA was replaced with non-complementary H7 target DNA. With the demonstrated superior sensing capabilities, this miniaturized CMOS sensing platform shows great potential for label-free point-of-care biosensing applications.     

MWCNT-ruthenium oxide composite paste electrode as non-enzymatic glucose sensor

A non-enzymatic glucose sensor of multi-walled carbon nanotube-ruthenium oxide/composite paste electrode (MWCNT-RuO(2)/CPE) was developed. The electrode was characterized by using XRD, SEM, TEM and EIS. Meanwhile, cyclic voltammetry and amperometry were used to check on the performances of the MWCNT-RuO(2)/CPE towards glucose. The proposed electrode has displayed a synergistic effect of RuO(2) and MWCNT on the electrocatalytic oxidation of glucose in 3M NaOH. This was possible via the formation of transitions of two redox pairs, viz. Ru(VI)/Ru(IV) and Ru(VII)/Ru(VI). A linear range of 0.5-50mM glucose and a limit of detection of 33μM glucose (S/N=3) were observed. There was no significant interference observable from the traditional interferences, viz. ascorbic acid and uric acid. Indeed, results so obtained have indicated that the developed MWCNT-RuO(2)/CPE would pave the way for a better future to glucose sensor development as its fabrication was without the use of any enzyme.     

Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results.     

Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.     

Sensitive and selective assay for fentanyl using gas chromatography with mass selective detection

A modified gas chromatographic assay, using mass-selective detection, has been developed for the quantitation of fentanyl in swine serum. Fentanyl and sufentanil, the internal standard, were extracted using a single-step liquid-liquid extraction with dichloromethane. Sensitivity and selectivity were improved by using electron-impact ionization (EI) in the selected-ion monitoring (SIM) mode, where fentanyl and sufentanil were monitored using the fragment ions at m/z 245 and 289, respectively. The limit of quantitation (LOQ) is 0.05 ng/ml, using 1 ml of sample, with a C.V. of 10.8% and a signal-to-noise ratio of 29. Standard curves were linear (r² = 0.999) over the working range of 0.05–1.5 ng/ml, using 1/y² as a weighting factor. Recoveries averaged 69.8 ± 4.7%, 91.0 ± 13.0% and 90.9 ± 10.3% at serum concentrations of 1.5, 0.5 and 0.1 ng/ml, respectively. Intra- and inter-day variances, were <12% at 0.1 ng/ml, and <10% at concentrations of 0.5, 1 and 1.5 ng/ml. Bias was 6.2% at the LOQ and ⩽12.8% at every other standard curve concentration. Applicability of the assay is demonstrated for the pharmacokinetic study of transdermally administered fentanyl in a postoperative swine.     

A novel NiO-Au hybrid nanobelts based sensor for sensitive and selective glucose detection

A novel amperometric nonenzymatic glucose sensor based on Au-doped NiO nanobelts has been successfully fabricated and applied to nonenzymatic glucose detection. Its electrochemical behavior towards the oxidation of glucose was compared with NiO nanofibers and Au microparticles prepared with a similar procedure. The NiO-Au hybrid nanobelts modified electrode displays greatly enhanced electrocatalytic activity towards glucose oxidation, revealing a synergistic effect between the matrix NiO and the doped Au. The as-prepared NiO-Au nanobelts based glucose sensor displays significantly lower onset potential, lower detection limit, higher sensitivity, and wider linear range than that of pristine NiO nanofibers modified electrode. Moreover, Au nanoparticles distributed in NiO nanofibers enabled amperometric glucose detection with insignificant interference from ascorbic acid and uric acid. These results indicate that the NiO-Au hybrid nanobelt is a promising candidate in the development of highly sensitive and selective nonenzymatic glucose sensors.     

Ultrasensitive detection of cellular protein interactions using bioluminescence resonance energy transfer quantum dot-based nanoprobes

Sensitive detection of protein interactions is a critical step toward understanding complex cellular processes. As an alternative to fluorescence-based detection, Renilla reniformis luciferase conjugated to quantum dots results in self-illuminating bioluminescence resonance energy transfer quantum dot (BRET-Qdot) nanoprobes that emit red to near-infrared bioluminescence light. Here, we report the development of an ultrasensitive technology based on BRET-Qdot conjugates modified with streptavidin ([BRET-Qdot]-SA) to detect cell-surface protein interactions. Transfected COS7 cells expressing human cell-surface proteins were interrogated with a human Fc tagged protein of interest. Specific protein interactions were detected using a biotinylated anti-human Fc region specific antibody followed by incubation with [BRET-Qdot]-SA. The luciferase substrate coelenterazine activated bioluminescence light emission was detected with an ultra-fast and -sensitive imager. Protein interactions barely detectable by the fluorescence-based approach were readily quantified using this technology. The results demonstrate the successful application and the flexibility of the BRET-Qdot-based imaging technology to the ultrasensitive investigation of cell-surface proteins and protein-protein interactions.     

Capacitive detection of glucose using molecularly imprinted polymers

A novel glucose biosensor based on capacitive detection has been developed using molecularly imprinted polymers. The sensitive layer was prepared by electropolymerization of o-phenylenediamine on a gold electrode in the presence of the template (glucose). Cyclic voltammetry and capacitive measurements monitored the process of electropolymerization. Surface uncovered areas were plugged with 1-dodecanethiol to make the layer dense, and the insulating properties of the layer were studied in the presence of redox couples. The template molecules and the nonbound thiol were removed from the modified electrode surface by washing with distilled water. A capacitance decrease could be obtained after injection of glucose. The electrode constructed similarly but with ascorbic acid or fructose only showed a small response compared with glucose. The stability and reproducibility of the biosensor were also investigated.     

Data registration and selective single-molecule analysis using multi-parameter fluorescence detection

A general strategy to identify and quantify sample molecules in dilute solution employing a new spectroscopic method for data registration and specific burst analysis denoted as multi-parameter fluorescence detection (MFD) was recently developed. While keeping the experimental advantage of monitoring single molecules diffusing through the microscopic open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence correlation spectroscopy, MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the four-dimensional fluorescence information (intensity, F; lifetime, tau; anisotropy, r; and spectral range, lambda(r)) in real time and allows for exclusion of extraneous events for subsequent analysis. In this review, the versatility of this technique in confocal fluorescence spectroscopy will be presented by identifying freely diffusing single dyes via their characteristic fluorescence properties in homogenous assays, resulting in significantly reduced misclassification probabilities. Major improvements in background suppression are demonstrated by time-gated autocorrelation analysis of fluorescence intensity traces extracted from MFD data. Finally, applications of MFD to real-time conformational dynamics studies of fluorescence labeled oligonucleotides will be presented.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.