Single-stranded Probes Research Articles

Methodological Research on Rapid Detection and Genotyping of NPM1 Gene Mutations in Leukemia

To establish a method for rapid detection and typing of NPM1 mutations in acute myeloid leukemia (AML) by fluorescence melting curve analysis technology. A pair of primers and a fluorescent single-stranded probe (molecule beacon) were designed for the mutant genes mutA, mutB, mutD in exon 12 of nucleopsin (NPM1) and wild type. With a real-time qPCR, the A, B, and D gene mutations of NPM1 were detected and typed by different-melting curve peak value of the probe through RT-PCR. This method could detected the mutations of A, B, and D in NPM1 effectively with a sensitivity of 1%. Furthermore, 62 AML clinical samples were evaluated by the method. In the results, the detection rate and typing of NPM1 mutations were consistent with the sequencing results of clinical samples. There are three features in the method of fluorescence melting curve analysis: stable PCR system, easy to operate, and the easily distinguishable results. The method might meet the demand for rapid typing of NPM1 gene mutation in early diagnosis or concomitant diagnosis of AML.

Multichannel Paper Chip-Based Gas Pressure Bioassay for Simultaneous Detection of Multiple MicroRNAs.

Simultaneous detection of multi-biomarkers not only enhances the accuracy of disease diagnosis but also improves detection efficiency and reduces cost. It is vital to achieve portable, simple, low-cost, and simultaneous detection of biomarkers for point-of-care (POC) diagnostics in a low-resource setting. Herein, a multichannel paper chip-based gas pressure bioassay was developed for the simultaneous detection of multiple biomarkers by combining multichannel paper chips with a portable gas pressure meter. Four DNA tetrahedral probes (DTPs) were used as capture probes and were immobilized in different detection zones of the paper chips to improve hybridization efficiency and reduce nonspecific adsorption. The formation of a sandwich structure between target microRNAs (miRNAs), the capture probe, and platinum nanoparticles (PtNPs)-modified complementary DNA (PtNPs-cDNA) transformed biomolecular recognition into quantitative detection of gas pressure. Four lung cancer-related miRNAs were detected simultaneously by a portable gas pressure meter. There is a good linear relationship between gas pressure and the logarithm of miRNA concentration in the range of 10 pM to 100 nM. Compared with single-stranded DNA capture probe, the signal-to-noise (S/N) of DNA tetrahedral probes improved more than 3 times. Using ring-oven washing, the unbound reagents in all channels of the paper chip were simultaneously and continuously washed away, leading to a more cheap, simple, and fast separation than magnetic separation. Therefore, it offers a promising multichannel paper chip-based gas pressure bioassay for portable and simultaneous detection of multiple biomarkers.

Electrospun Mn₂O₃ Nanofiber Networks as Bio-Transducers: Electrical Characterization, Modeling, and DNA Sensing

In this work, we report the synthesis and electrical characterization of electrospun manganese III oxide (Mn2O3) nanofibers and their application in chemiresistive biosensing. Here, the Mn2O3 nanofibers, which are inherently p-type semiconductors with a direct bandgap in the order of 1.2–1.4 eV, are drop cast across interdigitated electrodes to create the desired chemiresistive networks. Their I–V characteristics are governed by space charge limited current, attributing to nonlinear behavior at higher voltages. However, in the range of ±0.5 V, near-linear behavior is observed. Here, we have used an analogous R–C network to explain the low- and high-frequency behavior of the said nanofibers under different applied-bias conditions. Furthermore, the effect of temperature on the said nanofibers’ conductive properties is investigated, and the corresponding Arrhenius activation energy is derived. We have also explored the conductance–temperature dependence concerning the nanofiber network and have analyzed the potential carrier transport mechanisms. Also, operating in the near-linearity window, we have developed a chemiresistive protocol for DNA sensing. Mn2O3 nanofibers decorated with single-stranded probe DNAs act as transducers for surface hybridization with target nucleotides. The DNA sensing carried out in this work has a dynamic concentration range of 10 fM to 10 nM, with a linear response between 100 pM and 10 nM.

Activation of palindromes on a degradable modular grafting probe enables ultrasensitive detection of microRNAs.

This work describes a single-stranded degradable modular grafting probe for analyzing microRNA-21. In the system, the exonuclease activity of phi29 polymerase restrains the SYBR Green I/ssDNA induced background. The palindrome activation caused remarkable target fluorescence. The detection limit was achieved as 0.26 fM, showing potential in biochemical analysis.

Label-Free in-Situ Detection for DNA Hybridization Employing Grapefruit-Microstructured-Optical-Fiber-Based Microfluidic Whispering Gallery Mode Resonator

A whispering gallery mode (WGM) microresonator based on grapefruit microstructured optical fibers (MOFs) is proposed for in-situ DNA biosensing. The MOF microresonator has a porous structure that offers natural microfluidic channels to load microfluidic samples of volume down to 100 μL. Moreover, the WGM mechanism greatly enhances the interaction between WGM light and liquid sample kept inside the microcavity. The inner surface of the microcavity is functionalized with poly-L-lysine monolayer to achieve label-free binding of the single-strand probe DNA (ssDNA) through electrostatic force, and the probe immobilization as well as DNA hybridization processes are monitored in real time. The linear fitting of resonance wavelength shift versus DNA concentration in logarithmic scale indicates that DNA detection is available from 0.01 fmol/μL to 100 fmol/μL. The proposed WGM microresonator biosensor possesses such desirable merits as ease of operation, fast response, label free method, and high specificity, making it a promising candidate for in-situ DNA detection and related biosensing applications.

Gold nanoparticle-assisted plasmonic enhancement for DNA detection on a graphene-based portable surface plasmon resonance sensor

The impact of different gold nanoparticle (GNP) structures on plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (SPR) sensor. Two distinct structures of gold nano-urchins (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single-stranded probe DNA (ssDNA) of Mycobacterium tuberculosis complex DNA. The two types of GNP-ssDNA mixture were adsorbed onto the FLG-coated SPR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of complementary single-stranded DNA, the hybridization process took place and gradually removed the probes from the graphene surface. From SPR sensor preparation, the annealing process of the Au layer of the SPR sensor effectively enhanced the FLG coverage leading to a higher load of the probe DNA onto the sensing interface. The FLG was shown to be effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with the SPR sensor, GNu, with its rough and spiky structures, significantly reinforced the overall DNA hybridization signal compared with GNr with smooth superficies, especially in capturing the probe DNA. The DNA hybridization detection assisted by GNu reached the femtomolar range limit of detection. An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of the graphene-based SPR sensor and GNu-assisted DNA detection provided the proof-of-concept for the possibility of tuberculosis disease screening using a low-cost and portable system to be potentially applied in remote or third-world countries.

Graphene-based Electrochemical Biosensor for Impedimetric Detection of miRNAs as Potential Cancer Biomarkers

MicroRNAs (miRNAs) have shown great potential to be used as biomarkers for the screening and clinical diagnosis of cancer. In this study, an electrochemical biosensor based on graphene-modified glassy carbon electrode was developed for the detection of miRNA-21, a well-known biomarker for the early stage of prostate cancer. A novel molecular tethering agent was used for immobilization of single-stranded probe DNA onto the electrode surface. Different parameters related to biosensor fabrication and experimental conditions were optimized to obtain the highest biosensor response. Electrochemical impedance spectroscopy was employed to achieve a sensitive and label-free detection method for miRNA-21 through measuring the change in charge-transfer resistance (Rct) before and after hybridization. Under the optimal conditions, the biosensor showed a linear impedimetric response between ΔRct and logarithm of miRNA-21 concentration ranging from 10−14 to 10−8 M with a correlation coefficient of 0.972 and a detection limit of 3 fM. The selectivity of the biosensor was examined against non-complementary miRNA-141. The biosensor showed acceptable reproducibility, regeneration ability and stability as well as remarkable response (recoveries 90%–116%) in real plasma samples. The results indicated that the proposed biosensor could be used as a promising alternative to conventional methods in early clinical and point-of-care cancer diagnosis.

TFBG-SPR DNA-Biosensor for Renewable Ultra-Trace Detection of Mercury Ions

In this article, a tilted fiber Bragg grating (TFBG) plasmonic sensor based on thymine (T) - Hg - thymine (T) (T-Hg-T) base asymmetric pairing and Au nanoparticles (AuNPs), which can be used for highly sensitive and selective ultra-low-concentration Hg <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> detection, has been demonstrated. As is known, TFBG can provide an effective mean to easily excite the surface plasmon resonance (SPR) phenomenon while the fiber is coated with metal layer. By measuring the evolution of spectra, the SPR properties hence the surface perturbation could be detected accurately. To achieve highly sensitive and selective Hg <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> detection, the two specially designed single-strand probe DNAs (ssDNAs) with rich T base that can specifically forming stable T-Hg-T asymmetric pairing are served as a binding element for AuNPs labelled-target DNA conjugates. In the present of Hg <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> , these two ssDNAs that modified respectively on the surface of the sensor and the AuNPs will create a double helix T-Hg-T structure between the two DNA oligonucleotides and result in the surface refractive index perturbation amplified by the AuNPs. To overcome the low reusability of the AuNPs based DNA biosensor, by using iodide ions that can form more stable compounds with mercury ions over the T-Hg <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> -T structure, a regenerable DNA biosensor that can repeatedly detect mercury ions is developed. The experimental results demonstrate that this sensor shows an excellent ultra-low-concentration and selectivity detection capability for Hg <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> with the limit of detection (LOD) of 3.073 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-12</sup> M (3.073 pM) as well as a large dynamic range of 8 order of magnitude from 1.0 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-11</sup> M to 1.0 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-3</sup> M simultaneously. Thus, the experimental results in various actual sample from ultrapure/tap water to even clinical human serum, also reveal that it has potential application for Hg <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> detection in environmental samples and in clinic biological samples.

Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.

Enzyme- and label-free fluorescence microRNA biosensor based on the distance-dependent photoinduced electron transfer of DNA/Cu nanoparticles

Herein, we report a convenient enzyme- and label-free fluorescent biosensor for the quantitative detection of microRNA (miRNA). It is the first time that the distance-dependent property of photoinduced electron transfer (PET) has been observed between G-quadruplex/hemin (G/hemin) complexes and fluorescent DNA/Cu nanoparticles (NPs), accompanied by the change of the fluorescence intensity from DNA/Cu NPs. This innovative PET system could achieve the sensitive and specific determination of nucleic acids such as DNA and RNA based on the design of versatile detection sequences. With the existence of the target miRNA, the original flexible single-strand DNA detection probe becomes rigid DNA/RNA heteroduplexes. Therefore the PET could be hindered because of the prolonged distance from the electron donor-Cu NPs to electron acceptor-G/hemin complexes. Hence, a fluorescence light-up miRNA biosensor was established via the monitoring of the fluorescence signal of the formed DNA/Cu NPs. Compared to other reported PET-based miRNA determination methods, the distance-dependent PET of Cu NPs-based biosensor only needs to adjust the detection probe sequences for various miRNAs assays. This strategy is effective for the determination of the miRNA let-7a over a linear range of 0.5 to 100 nM, with a relatively low limit of detection of 0.2 nM. Besides, this novel assay has been successfully employed for the enzyme- and label-free fluorescence detection of miRNA from real samples.

Experimental and theoretical study for miR-155 detection through resveratrol interaction with nucleic acids using magnetic core-shell nanoparticles.

A novel electrochemical nanobiosensor for the detection of miR-155 (as breast cancer biomarker) is introduced . Fe3O4NPs@Ag core-shell nanoparticles were synthesized and their shape and characteristics were confirmed by scanning electron microscope (SEM) imaging, Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) methods. Synthesized nanoparticles were applied onto the magnetic bar carbon paste electrode and then the amine-modified anti-miR-155 (single-stranded probes) was applied on the modified electrode surface and upon hybridization with target miR-155, resveratrol (RSV) was eventuallyapplied as an electrochemical label on the double-strand oligonucleotide. Differential pulse voltammetry (DPV) of the oxidation peak of RSV was assumed as the final signal by sweeping potential from 0 to 0.6V (vs. Ag/AgCl). The fabrication process was optimized through a series of experiments and the optimized process was confirmed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Thelinear range of the fabricated nanobiosensor was 0.5fM to 1.0nM and the detection limit was 0.15fM. The nanobiosensor was able to pass reproducibility and specificity tests using different types of mismatched target sequences.Spiked real samples of human serum were used to confirm thatthe nanobiosensor enablesdetection of miR-155 without any significant interferences from other moieties and molecules. Finally, the molecular dynamics simulation of the RSV interaction with single- and double-stranded oligonucleotide was performed and confirmed the preferential binding ofRSV to double-stranded DNA; therefore, it can be used as the electrochemical label of DNA and/or miRNA hybridization-based biosensors. Graphical abstract.

Graphene oxide-based molecular diagnostic biosensor for simultaneous detection of Zika and dengue viruses

The world’s tropical and sub-tropical regions are at risk for Zika and dengue virus (ZIKV, DENV) spread. Co-infection between ZIKV and DENV and repeated DENV infections of different serotypes increase the disease severity by impairing immune responses. Therefore, to avoid high risks of virus infection and achieve effective clinical management, development of diagnostic sensor for rapid and accurate identification of each virus is very important. Herein, we report a graphene oxide-based multiplexed detection strategy to make the differential diagnosis of Zika and dengue viruses by targeting virus nucleic acid. The fluorometric detection strategy is integrated with loop-mediated isothermal amplification assay to further increase sensitivity. In the presence of target virus, fluorescent PNA probes recognize the single-stranded loops of the cauliflower-like-shaped virus amplicon in a sequence-specific manner. Meanwhile, graphene oxide functions to distinguish the valid fluorescence signal as a selective fluorescence quencher for single-stranded PNA probes. The present strategy allows multiplexed detection of closely related viruses for specific discrimination of the virus nucleic acids with simplified molecular diagnostic steps, without requiring any nucleic acid isolation step.

High-coverage SARS-CoV-2 genome sequences acquired by target capture sequencing.

In this study, we designed a set of SARS‐CoV‐2 enrichment probes to increase the capacity for sequence‐based virus detection and obtain the comprehensive genome sequence at the same time. This universal SARS‐CoV‐2 enrichment probe set contains 502 120 nt single‐stranded DNA biotin‐labeled probes designed based on all available SARS‐CoV‐2 viral sequences and it can be used to enrich for SARS‐CoV‐2 sequences without prior knowledge of type or subtype. Following the CDC health and safety guidelines, marked enrichment was demonstrated in a virus strain sample from cell culture, three nasopharyngeal swab samples (cycle threshold [C t] values: 32.36, 36.72, and 38.44) from patients diagnosed with COVID‐19 (positive control) and four throat swab samples from patients without COVID‐19 (negative controls), respectively. Moreover, based on these high‐quality sequences, we discuss the heterozygosity and viral expression during coronavirus replication and its phylogenetic relationship with other selected high‐quality samples from the Genome Variation Map. Therefore, this universal SARS‐CoV‐2 enrichment probe system can capture and enrich SARS‐CoV‐2 viral sequences selectively and effectively in different samples, especially clinical swab samples with a relatively low concentration of viral particles.

Aptamer based high throughput colorimetric biosensor for detection of staphylococcus aureus

To develop a high throughput colorimetric biosensor for detection of Staphylococcus aureus (SA) based on specific aptamer and catalysis of dsDNA-SYBR Green I (SG I) complex. SA specific aptamer was immobilized on a 96-well plate by hybridization with the capture probe anchored on the plate surface through streptavidin-biotin binding. In presence of SA, the aptamer was dissociated from the capture probe-aptamer duplex due to the stronger interaction between the aptamer and SA. The consequent single-strand capture probe could be hybridized with a three-way junction (TWJ) probe. With the presence of SG I, the dsDNA-SG I complex catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) under photo-irradiation, producing sensitive photo-catalyzed colorimetric response to SA. Under the optimal conditions, the proposed method could directly detect SA with the limit of detection (LOD) at 81 CFU mL−1 in PBS buffer in 5.5 hours, which demonstrated the sensitive and fast quantification of target pathogenic bacteria. The method showed weak colorimetric signal to Escherichia coli and Pseudomonas aeruginosa, indicating the high specificity for SA. In addition, the method can simultaneously detect 96 samples which can be used for high throughput analysis. The designed method may become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety and environmental monitoring.

Label-Free Electrochemical Detection of DNA Hybridization: A Method for COVID-19 Diagnosis.

This paper presents label-free electrochemical transduction as a suitable scheme for COVID-19-specific viral RNA/c-DNA detection, with an aim to facilitate point of care diagnosis. In lieu of this, we discuss the proposed electrochemical biosensing scheme, based on electrodeposited gold nanoparticles as the transducing elements. Specific to this approach, here, the protocols associated with the immobilization of the single-stranded probe nucleotide on to the biosensor, have also been laid out. This paper also discusses the methods of electrochemical analysis, to be used for data acquisition and subsequent calibration, in relation to target analyte detection. Towards facilitating portable diagnosis, development of miniaturized sensors and their integration with readout units have also been discussed.

Construction of hybrid DNAs@CP for the rapid synchronous sensing of multiplex microRNAs based on experimental studies and molecular simulation

A water stable one-dimensional (1D) ladder-shaped coordination polymer (CP) has been synthesized and exhibits a strong affinity to two fluorescein-tagged single-stranded probe DNAs (P-DNAs), giving a sensing platform of P-DNAs@1. Such a hybrid sensing platform is capable of simultaneous detection of breast cancer related microRNA-221 (miRNA-221) and miRNA-222 in a specific and synchronous manner, without observable cross-reactions, as supported by experimental evidences. The interaction mode and the electronic energy between CP 1 with nucleic acid were confirmed by molecular simulation and the universal force field (UFF).

Multi-cycle signal-amplified colorimetric detection of tobramycin based on dual-strand displacement and three-way DNA junction

A colorimetric aptasensor for tobramycin was developed using a multi-cycle signal amplification strategy which combines the application of dual-strand displacement and DNA three-way junction structure with the catalysis of G-quadruplex/hemin DNAzyme. The assay starts with a partially complementary double-stranded probe template1/template2 (T1/T2). The aptamer of tobramycin is included in T1. Tobramycin can bind with the aptamer region of T1 and expose the 3′-terminal of T2. In the presence of a pair of primers, Bsm DNA polymerase and Nt.BstNBI endonuclease-assisted isothermal amplification generates a single-stranded DNA reporter probe. The later can hybridize with a specially designed DNA three-way junction and further trigger Lambda exonuclease-assisted loop amplification, which regenerates the reporter probe and produces a large number of signal probe (containing G-quadruplex-forming sequence). Finally, the G-quadruplex/hemin DNAzyme catalyzes ABTS2−/H2O2 reaction system to generate a colorimetric signal. The assay exhibits high selectivity and sensitivity to tobramycin, with a detection limit of 12.24 nM and a linear response range of 20–800 nM. Moreover, the assay was successfully used to detect tobramycin in milk and water samples, indicating its potential application in both food safety control and environmental monitoring.

Ferrocene-labeled and purification-free electrochemical biosensor based on ligase chain reaction for ultrasensitive single nucleotide polymorphism detection

Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A′, and B′, where B′ is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A′B′ in 2n. Subsequently, immobilization of the Fc-tagged duplex AB-A′B′ on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A′B′, such strategy realized directly analysis of LCR products without the need for purification. By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM–10 pM), and the recovery rates (95.3%–107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.

A highly sensitive electrochemical biosensor for protein based on a tetrahedral DNA probe, N- and P-co-doped graphene, and rolling circle amplification

A tetrahedral DNA probe can effectively overcome the steric effects of a single-stranded probe to obtain well-controlled density and minimize nonspecific adsorption. Herein, a highly sensitive electrochemical biosensor is fabricated for determination of protein using a tetrahedral DNA probe and rolling circle amplification (RCA). N- and P-co-doped graphene (NP-rGO) is prepared, and AuNPs are then electrodeposited on it for DNA probe immobilization. Benefitting from the synergistic effects of the excellent electrical conductivity of NP-rGO, the stability of the tetrahedral DNA probe and the signal amplification of RCA, the biosensor achieves a low limit of 3.53 × 10-14M for thrombin and a wide linear range from 1 × 10-13 to 1 × 10-7M. This study provides a sensitive and effective method for the detection of protein in peripheral biofluids, and paves the way for future clinical diagnostics and treatment of disease. Graphical abstract.

Synthesis of Single-Stranded RNA Probes by In Vitro Transcription.

In this protocol, we describe procedures for synthesizing RNA probes of high specific activity from DNA templates containing promoters for bacteriophage-encoded RNA polymerases. The DNA fragment to be transcribed should have previously been cloned into one of the commercially available plasmids containing bacteriophage RNA polymerase promoters. Alternatively, DNA templates can be synthesized in polymerase chain reactions (PCRs) using gene-specific primers whose 5' ends encode synthetic promoters for bacteriophage-encoded RNA polymerases.