Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.

An electrochemiluminescence aptasensor based on highly luminescent silver-based MOF and biotin-streptavidin system for mercury ion detection.

In this study, for the first time, a silver-based metal-organic framework (Ag-MOF) was synthesized and used as the electrochemiluminescence (ECL) emitter for building an ECL sensor. After modification with chitosan (CS) and gold nanoparticles (Au NPs), the ECL stability of Ag-MOF was improved. To detect mercury ions, a biosensor was constructed using the mercury ion aptamer and steric effect of streptavidin. First, the capture strand (cDNA) with terminal-modified sulfhydryl group was attached to the electrode surface by the Au-S bond. Then, the mercury-ion aptamer (Apt-Hg) modified with biotin was anchored to the electrode by complementary pairing with cDNA. Streptavidin (SA) could be fixed on the electrode by linking with biotin, thereby reducing the ECL signal. However, in the presence of mercury ions, the aptamer was removed and streptavidin could not be immobilized on the electrode. Hence, the ECL signal of the sensor increased with the concentration of mercury ions, which was linear in the range from 1 µM to 300 fM. The detection limit could reach 66 fM (S/N = 3). The sensor provided a new method for the detection of mercury ions.

Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared.

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Oligonucleotide-Blocked Streptavidin for Biotinylation Analysis.

Binding between streptavidin, or its homologues, to biotin is one of the most widely exploited biological interactions in the biomedical sciences. Controlling the extent of biotinylation is important for meeting the requirements of the intended design and to preserve the native function of the biotin recipient. Within the protein world, a"trial-and-error" optimization approach toward biotinylation reaction conditions is often necessary due to widely varying properties of proteins. Therefore, product analysis is important. We show here that a oligonucleotide-blocked streptavidin, effectively "monovalent streptavidin", can tag biotin moieties individually and the tagged products visualized via a polyacrylamide gel shift assay to reveal the product distribution, i.e., [protein-(biotin)n] products where n = 1, 2, 3, etc. This is in contrast, and complementary, to current commercially available analytical reagents for biotinylation characterization, which use an absorbance or fluorescence signal to yield the mean number of biotin moieties.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (S/N) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high S/N ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes. Second, fluorescent-encoded beads (FEBs), coupled with the corresponding target sequence-specific capture probes through streptavidin-biotin conjugation, are used to partition amplicons via hybridization according to the Poisson distribution. Finally, FEBs mixed with digital PCR mixes are isolated into droplets generated via Sapphire chips (Naica Crystal Digital PCR system) to complete the digital PCR and result analysis. For proof of concept, we demonstrate that this method achieves high S/N ratios in a 5-plexed assay for influenza viruses and SARS-CoV-2 at concentrations below 10 copies and even close to a single molecule per reaction without cross-reaction, further verifying the possibility of clinical actual sample detection with 100% accuracy, which paves the way for the realization of digital PCR with ultrahigh coding capacity and ultra-sensitivity.

Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis.

The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5'-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules.

Prevalence of Detectable Biotin in Five US Emergency Department Patient Cohorts.

BACKGROUND: The objective of this study was to estimate the prevalence of biotin supplementation in United States emergency department patients using a multi-site, geographically distributed sampling model. METHODS: Biotin was measured using an Abbott ARCHITECT Biotin research use only assay in 7118 emergency department patient serum or plasma samples from five US medical centers. Samples with biotin ≥10 ng/mL underwent additional LC-MS/MS confirmatory testing for biotin and its primary metabolites. The overall and site-specific prevalence of detectable biotin was determined using the screening assay while biotin speciation (i.e., prevalence of detectable metabolites) was determined using LC-MS/MS. RESULTS: Of 7118 samples screened, 291 (4.1%) had biotin ≥10 ng/mL and were considered positive. Across five medical centers, the fraction of positive samples ranged from 2.0% to 5.4%. The maximum biotin concentration observed was 355 ng/mL. Of the 285 positive screens that underwent additional LC-MS/MS testing, 89 (31%) showed detectable biotin, bisnorbiotin, and/or biotin sulfoxide. Biotin, bisnorbiotin, and biotinsulfoxide were detected in 82/89 (92.1%), 61/89 (68.5%), and 18/89 (20.2%) samples, respectively; biotin was detected in the absence of either metabolite in 18/89 (20.2%) samples. CONCLUSIONS: Using a screening assay, 4.1% of emergency department patient samples were found to be potentially susceptible to interference from biotin. Confirmatory testing showed detectable biotin and/or biotin metabolites in 31% of positive screens (1.3% overall). The prevalence of biotin ≥10 ng/mL varied 2-3-fold across US emergency department patient cohorts. Biotin metabolites were observed in 80% of samples confirmed to have detectable biotin species by LC-MS/MS, suggesting that rigorous assessments of assay susceptibility to biotin interference, often performed using in vitro studies, should consider the potential role of biotin metabolites present in vivo.

Biotin immunoassay interference: A UK-based prevalence study.

BACKGROUND: Biotin interference in biotin-streptavidin-based immunoassays is increasingly reported due to individuals taking biotin-containing supplements and patients prescribed biotin. The reported prevalence of serum biotin above the lowest threshold (≥10 µg/L) for interference in Roche Diagnostics immunoassay tests is 0.8% in Australia and 7.4% in the USA. There are, however, no such data in UK populations. In a service evaluation, we therefore studied the prevalence of biotin interference in routine serum samples received in our laboratory. METHODS: Biotin was measured in 524 anonymized surplus serum samples in which at least one immunoassay test had been requested. RESULTS: The median (95% confidence intervals) for serum biotin was 0.27 µg/L (0.07-0.93 µg/L). Serum biotin was <10 µg/L in all samples, <5 µg/L in 522 (99.6%) and <1 µg/L in 513 (98.1%) samples. In four samples, serum biotin was ≥2.5 µg/L (0.8%). CONCLUSIONS: These data indicate that the probability of biotin immunoassay interference in our patient population is extremely low, with the exception of assays reporting the lowest interference thresholds (e.g. Ortho Troponin I assay [threshold ≥2.5 µg/L]).

Biotinylated Streptavidin Surface Coating Improves the Efficacy of a PLGA Microparticle-Based Cancer Vaccine.

Triple-negative breast cancer (TNBC) is an immune-enriched subset of breast cancer that has recently demonstrated clinical responsiveness to combinatorial immunotherapy. However, the lack of targeted interventions against hormone receptors or HER2 continues to limit treatment options for these patients. To begin expanding available interventions for patients with metastatic TNBC, we previously reported a therapeutic vaccine regimen that significantly reduced spontaneous lung metastases in a preclinical TNBC model. This heterologous vaccine approach "primed" mice with tumor lysate antigens encapsulated within poly(lactic-co-glycolic) acid microparticles (PLGA MPs), and then "boosted" mice with tumor lysates plus adjuvant. The use of the PLGA MP prime as monotherapy demonstrated no efficacy, suggesting that improving this component of our therapy would achieve greater vaccine efficacy. Here, we functionally improved the PLGA MP prime by coating microparticles with biotinylated streptavidin-conjugated using 1-ethyl-3-(3-dimethylaminoproplyl) carbodiimide/N-hydroxysuccinimide (EDC/Sulfo-NHS) linkers. This modification enhanced the immunostimulatory potential of our PLGA MPs, as evidenced by increased phagocytosis, maturation, and stimulatory ligand expression by antigen-presenting cells (APCs). Therapeutic prime/boost vaccination of TNBC-bearing mice with surfaced-coated PLGA MPs significantly reduced spontaneous lung metastases by an average of 56% relative to mice primed with unmodified PLGA MPs, and a significant 88% average reduction in spontaneous lung metastases relative to untreated control mice. These findings illustrate that relatively common biotin-streptavidin conjugation formulations can positively affect microparticle-based vaccine immunogenicity resulting in enhanced therapeutic efficacy against established preclinical mammary tumors.

Enantioselective Hydroxylation of Benzylic C(sp3)-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology.

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.

Competitive HRP-Linked Colorimetric Aptasensor for the Detection of Fumonisin B1 in Food based on Dual Biotin-Streptavidin Interaction.

Fumonisin B1 (FB1) is the most prevalent and toxic form among fumonisin homologues which are produced by fusarium species and it contaminates various types of food products, posing serious health hazards for humans and animals. In this work, a colorimetric assay for the detection of FB1 has been developed based on competitive horseradish peroxidase (HRP)-linked aptamer and dual biotin-streptavidin interaction. In short, a biotinylated aptamer of FB1 was immobilized on the microplate by biotin-streptavidin binding; the complementary strand (csDNA) of the aptamer was ligated with HRP by biotin-streptavidin binding again to form a csDNA-HRP sensing probe, competing with FB1 to bind to the aptamer. The color change can be observed after the addition of chromogenic and stop solution, thereby realizing the visual detection of FB1. Under optimal conditions, good linearity was observed within the concentration range of 0.5 to 300 ng/mL, with a detection of limit of 0.3 ng/mL. This assay is further validated by spike recovery tests towards beer and corn samples, it provides a simple, sensitive and reliable method for the screening of FB1 in food samples and may be potentially used as an alternative to conventional assays.

A strategy for iron oxide nanoparticles to adhere to the neuronal membrane in the substantia nigra of mice.

Effective attachment of magnetic nanoparticles to neuronal membranes has far-reaching significance in activating ion channels and treating neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) synthesized by the polyol pyrolysis method have the advantages of rich surface functional groups, excellent magnetic properties, controllable particle size and water dispersibility. We propose that perfusion of biotin into the targeted brain area should be initially performed because it tends to be adsorbed by cell membranes, followed by injection of streptavidin (SA)-modified SPIONs into the same area of the brain. By means of the strong binding force between SA and biotin, the SPIONs may subsequently adhere to the cell surfaces in the brain area. In this work, fluorescein isothiocyanate-streptavidin (FITC-SA) was modified on the surface of polyethylene imine (PEI)-SPIONs by the EDC-NHS method and stereotaxically injected into the biotin-supplemented substantia nigra of mice. The combination of fluorescence detection with transmission electron microscopy (TEM) confirmed that FITC-SA/PEI-SPIONs adhered to neuronal membranes in the substantia nigra of mice 24 h after injection. The results show that our strategy can promote the attachment of SPIONs to neuronal membranes.

Characterization of biotin interference in 21 Vitros 5600 immunoassays and risk mitigation for patient safety at a large academic medical center.

BACKGROUND: Biotin and streptavidin are commonly used reagents in clinical immunoassays. Several cases of biotin interference with immunoassay testing for patients taking biotin supplements have been reported, yet, not all analytes and platforms susceptible to biotin interference have been characterized. The objectives of this study are to characterize biotin interference with 21 immunoassays using the Ortho Clinical Diagnostics Vitros 5600, evaluate a biotin-depletion method, and apply risk mitigation strategies for biotin interference during routine clinical testing at our institution. METHODS: Residual serum without and with increasing concentrations of exogenous biotin were used to evaluate biotin interference with 21 immunoassays using the Vitros 5600. Biotin-depletion was evaluated by comparing measured analyte concentrations in serum with and without exogenous biotin and streptavidin-microparticle pretreatment. Focused education for healthcare professionals about biotin interference was performed in February 2018. Samples with suspected biotin interference were investigated using this biotin-depletion method, and analyte testing by alternate methodology for select samples. RESULTS: Exogenous biotin in serum caused dose-dependent negative biases in 15 immunometric assays, and dose-dependent positive biases in 6 competitive immunoassays. Streptavidin-microparticle pretreatment of serum containing exogenous biotin demonstrated recoveries 100 ±â€¯15% of expected values for all 21 analytes. Physicians identified 21 samples suspicious for biotin interference over 11 months, and streptavidin-microparticle pretreatment verified 11 cases of biotin interference. CONCLUSIONS: Analytical bias caused by biotin interference is dependent on biotin concentration but independent of analyte concentration for immunometric methods using the Vitros 5600, and dependent on both biotin and analyte concentration for competitive immunoassays. Multi-disciplinary education and a lab streptavidin-microparticle pretreatment method help mitigate risk of erroneous results due to biotin interference for patient safety.

Aptamer-Based Nanoporous Anodic Alumina Interferometric Biosensor for Real-Time Thrombin Detection.

Aptamer biosensors are one of the most powerful techniques in biosensing. Achieving the best platform to use in aptamer biosensors typically includes crucial chemical modifications that enable aptamer immobilization on the surface in the most efficient manner. These chemical modifications must be well defined. In this work we propose nanoporous anodic alumina (NAA) chemically modified with streptavidin as a platform for aptamer immobilization. The immobilization of biotinylated thrombin binding aptamer (TBA) was monitored in real time by means of reflective interferometric spectroscopy (RIfS). The study has permitted to characterize in real time the path to immobilize TBA on the inner pore walls of NAA. Furthermore, this study provides an accurate label-free method to detect thrombin in real-time with high affinity and specificity.

Aptamer Structure Switch Fluorescence Anisotropy Assay for Small Molecules Using Streptavidin as an Effective Signal Amplifier Based on Proximity Effect.

Fluorescence polarization/anisotropy (FP/FA) approaches are appealing for targets sensing in homogeneous solution due to simplicity, reproducibility and sensitivity. Taking advantage of aptamers, aptamer structure switch FA methods are unique for small molecule detection based on the competition between aptamer-target binding and the hybridization of aptamer and complementary DNA (cDNA). However, usually small FA change is generated in these aptamer assays that only rely on size change caused by hybridization of an oligonucleotide because of the rapid local rotation of fluorophores and small mass change. Here we describe a simple and general aptamer structure switch FA assay for small molecules by employing a large-sized streptavidin (SA) as an effective signal amplifier based on proximity effect to reduce local rotation of fluorophore. In this design, the SA-labeled cDNA hybridizes with fluorescein (FAM)-labeled aptamer, drawing FAM close to SA and bringing a much higher FA value due to restricted local rotation of FAM. Small molecule-aptamer probe binding causes displacement of the SA-labeled cDNA and great decrease of FA. The closeness of SA to FAM in the duplex is key for this proposed strategy to produce large FA changes in target detection. Our method enabled to detect 60 pM aflatoxin B1 (AFB1), 1 nM ochratoxin A (OTA), and 0.5 µM adenosine triphosphate (ATP), respectively. This aptamer FA method combines the merits of aptamers and FA analysis, and it is promising in applications of detection of small molecules with good sensitivity.

Human papilloma virus DNA-biomarker analysis for cervical cancer: Signal enhancement by gold nanoparticle-coupled tetravalent streptavidin-biotin strategy.

Human papillomavirus (HPV) is a double-standard DNA virus, as well as the source of infection to the mucous membrane. It is a sexually transmitted disease that brings the changes in the cervix cells. Oncogenes, E6 and E7 play a pivotal role in the HPV infection. Identifying these genes to detect HPV strains, especially a prevalent HPV16 strain, will bring a great impact. Among different sensing strategies for pathogens, the dielectric electrochemical biosensor shows the potential due to its higher sensitivity. In this research, HPV16-E7 DNA sequence was detected on the carbodiimidazole-modified interdigitated electrode (IDE) surface with the detection limit of 1 fM. To enhance the sensitivity, the target sequence was conjugated on gold nanoparticle (GNP) and attained detection to the level of 10 aM. This produced ~100 folds improvement in detecting HPV16-E7 gene and 4 folds increment in the current flow. The stability of HPV16-E7 DNA sequences on GNP was verified by the salt-induced GNP aggregation. The current system has shown the higher specificity by comparing against non-complementary and triple-mismatched DNA sequences of HPV16-E7. This demonstration in detecting HPV16-E7 using dielectric IDE sensing system with a higher sensitivity can be recommended for detecting a wide range of disease-causing DNA-markers.

Bioluminescent aptamer-based solid-phase microassay to detect lung tumor cells in plasma.

Two high-affinity DNA aptamers for lung tumor cells were applied as biospecific elements in bioluminescent assay of patient blood. The oligonucleotide complementary to the 5' end of both aptamers carrying either biotin or Ca2+-regulated photoprotein obelin was used to form a sandwich-type analytical complex on the surfaces of magnetic streptavidin-activated microspherical particles. Clinical blood samples from cases of morphologically confirmed lung cancer and control samples were analyzed applying the developed assay. From the receiver operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers the sensitivity of 91.5% and the specificity of 75% (p < 0.001). The area under ROC curve with the value of 0.901 distinguishes well between the two groups under investigation.

Biotin interference in immunoassays based on biotin-strept(avidin) chemistry: An emerging threat.

Biotinylated antibodies/antigens are currently used in many immunoassay formats in clinical settings for diversified analytes and biomarkers to offer high detection selectivity and sensitivity. Biotin cannot be synthesized by mammals and must be taken as an essential supplement. Normal intake of biotin from various foods and milk causes no effect on the streptavidin/biotin-based immunoassays. However, overconsumption of biotin (daily doses 100-300 mg) poses a significant problem for immunoassays using the biotin-strept(avidin) pair. Biotin interferences are noted in immunoassays of thyroid markers, drugs, hormones, cancer markers, the biomarker for cardiac function (ß-human chorionic gonadotropin), etc. The biotin level required for serious interference in test results varies significantly from test to test and cannot easily be predicted. Immunoassay manufacturers with technologies based on strept(avidin)-biotin binding must investigate the interference from biotin (up to at least 1200 ng/mL or 4.9 µM of biotin) in various formats. There is no concrete solution to circumvent the biotin interference encountered in blood samples, short of biotin removal. Considering the short half-life of biotin in the human body, patients must stop taking biotin supplements for >48 h before the test. However, this scenario is not considered for patients in emergency situations or those with biotinidase deficiency, mitochondrial metabolic disorders or multiple sclerosis. Apparently, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays using strep(avidin)-biotin chemistry. To date, there is no quick and reliable procedure for the detection of biotin at below nanomolar levels in blood and biological samples. Traditional lab-based techniques including HPLC/MS-MS cannot process an enormous number of public samples. Biosensors with high detection sensitivity, miniaturization, low cost, and multiplexing have the potential to address this issue.

Ultrasensitive amplification-free detection of protein kinase based on catalyzed assembly and enumeration of gold nanoparticles.

A single-particle enumeration method based on phosphorylation-directed in situ assembly of gold nanoparticles is developed for the ultrasensitive sensing of cellular protein kinase A activity. In comparison to existing strategies, the proposed new method demonstrates five orders of linear range and improves the detection limit up to 10-to-1000 fold without the involvement of target amplification.