PCR-free Detection Research Articles

Biporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection

Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.

PCR-Free Innovative Strategies for SARS-CoV-2 Detection.

The pandemic outbreak caused by SARS-CoV-2 coronavirus brought a crucial issue in public health causing up to now more than 600 million infected people and 6.5 million deaths. Conventional diagnostic methods are based on quantitative reverse transcription polymerase chain reaction (RT-qPCR assay) and immuno-detection (ELISA assay). However, despite these techniques have the advantages of being standardized and consolidated, they keep some main limitations in terms of accuracy (immunoassays), time/cost consumption of analysis, the need for qualified personnel, and lab constrain (molecular assays). There is crucial the need to develop new diagnostic approaches for accurate, fast and portable viral detection and quantification. Among these, PCR-free biosensors represent the most appealing solution since they can allow molecular detection without the complexity of the PCR. This will enable the possibility to be integrated in portable and low-cost systems for massive and decentralized screening of SARS-CoV-2 in a point-of-care (PoC) format, pointing to achieve a performant identification and control of infection. In this review, the most recent approaches for the SARS-CoV-2 PCR-free detection are reported, describing both the instrumental and methodological features, and highlighting their suitability for a PoC application.

DCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration.

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9's specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant.

Ultrasensitive PCR-Free detection of whole virus genome by electrochemiluminescence

Detection of nucleic acids is crucial in many medical applications, and in particular for monitoring infectious diseases, as it has become perfectly clear after the pandemic infection of COVID-19. In this context, the development of innovative detection methods based on signal-amplification rather than analyte-amplification represents a significant breakthrough compared to existing PCR-based methodologies, allowing the development of new nucleic acid detection technologies suitable to be integrated in portable and low-cost sensor devices while keeping high sensitivities, thus enabling massive diagnostic screening. In this work, we present a novel molecular sensor for the ultrasensitive PCR-free detection of Hepatitis B Virus (HBV) based on electrochemiluminescence (ECL). Thanks to the combination of surface cooperative hybridization scheme with ECL detection strategy, our novel DNA sensor is able to detect HBV genome – both synthetic and extracted – with the unprecedented limit of detection (LoD) of 0.05 cps μL−1 for extracted sample, that is even lower than the typical LoD of PCR methodologies. The detection concept presented here for HBV detection is very versatile and can be extended to other pathogens, paving the way for future development of rapid molecular test for infectious diseases, both viral and bacterial, in Point-of-Care (PoC) format.

A CRISPR/Cas12a-Mediated Dual-Mode Electrochemical Biosensor for Polymerase Chain Reaction-Free Detection of Genetically Modified Soybean.

A clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-mediated dual-mode electrochemical biosensor without polymerase chain reaction (PCR) amplification was designed for sensitive and reliable detection of genetically modified soybean SHZD32-1. A functionalized composite bionanomaterial Fe3O4@AuNPs/DNA-Fc&Ru was synthesized as the signal unit, while a characteristic gene fragment of SHZD32-1 was chosen as the target DNA (tDNA). When Cas12a, crRNA, and tDNA were present simultaneously, a ternary complex Cas12a-crRNA-tDNA was formed, and the nonspecific cleavage ability of the CRISPR/Cas12a system toward single-stranded DNA was activated. Thus, the single-stranded DNA-Fc in the signal unit was cleaved, resulting in the decrease in the fast scan voltammetric (FSV) signal from ferrocene (Fc) and the increase in the electrochemiluminescence (ECL) signal from ruthenium complex (Ru) inhibited by Fc. The linear range was 1-107 fmol/L for ECL and 10-108 fmol/L for FSV, and the limit of detection (LOD) was 0.3 fmol/L for ECL and 3 fmol/L for FSV. Accuracy, precision, stability, selectivity, and reliability were all satisfied. In addition, PCR-free detection could be completed in an hour at room temperature without requiring complicated operation and sample processing, showing great potential in the field detection of genetically modified crops.

Ultrasensitive Electrochemical Impedance Detection of Mycoplasma agalactiae DNA by Low-Cost and Disposable Au-Decorated NiO Nanowall Electrodes.

Nanostructured electrodes detecting bacteria or viruses through DNA hybridization represent a promising method, which may be useful in on-field applications where PCR-based methods are very expensive, time-consuming, and require trained personnel. Indeed, electrochemical sensors combine disposability, fast response, high sensitivity, and portability. Here, a low-cost and high-surface-area electrode, based on Au-decorated NiO nanowalls, demonstrates a highly sensitive PCR-free detection of a real sample of Mycoplasma agalactiae (Ma) DNA. NiO nanowalls, synthesized by aqueous methods, thermal annealing, and Au decoration, by electroless deposition, ensure a high-surface-area platform for successful immobilization of Ma thiolated probe DNA. The morphological, chemical, and electrochemical properties of the electrode were characterized, and a reproducible detection of synthetic Ma DNA was observed and investigated by impedance measurements. Electrochemical impedance spectroscopy (EIS) ascribed the origin of impedance signal to the Ma DNA hybridization with its probe immobilized onto the electrode. The electrode successfully discriminates between DNA extracted from healthy and infected sheep milk, showing the ability to detect Ma DNA in concentrations as low as 53 ± 2 copy number μL-1. The Au-decorated NiO nanowall electrode represents a promising route toward PCR-free, disposable, rapid, and molecular detection.

PCR-Free Detection of Long Non-Coding HOTAIR RNA in Ovarian Cancer Cell Lines and Plasma Samples.

Long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is one of the promising biomarkers that has widely been used in determining the stages of many cancers, including ovarian cancer. In cancer diagnostics, the two key analytical challenges for detecting long non-coding RNA biomarkers are i) the low concentration levels (nM to fM range) in which they are found and ii) the analytical method where broad dynamic range is required (four to six orders of magnitude) due to the large variation in expression levels for different HOTAIR RNAs. To meet these challenges, we report on a biosensing platform for the visual (colorimetric) estimation and subsequent electrochemical quantification of ovarian-cancer-specific HOTAIR using a screen-printed gold electrode (SPE-Au). Our assay utilizes a two-step strategy that involves (i) magnetic isolation and purification of target HOTAIR sequences and (ii) subsequent detection of isolated sequences using a sandwich hybridization coupled with horseradish peroxidase (HRP)-catalyzed reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. The assay achieved a detection limit of 1.0 fM HOTAIR in spiked buffer samples with excellent reproducibility (% RSD ≤ 5%, for n = 3). It was successfully applied to detect HOTAIR in cancer cell lines and a panel of plasma samples derived from patients with ovarian cancer. The analytical performance of the method was validated with standard RT-qPCR. We believe that the proof of concept assay reported here may find potential use in routine clinical settings for the screening of cancer-related lncRNAs.

Ultrasensitive THz biosensor for PCR-free cDNA detection based on frequency selective surfaces.

THz technologies are a powerful tool for label-free detection of biomolecules. However, significant reduction of the lower detection limit is required to apply THz-sensors in biomedical diagnosis. This paper reports an ultrasensitive THz-biosensor based on asymmetric double split ring resonators (aDSRR) for the direct label- and PCR-free detection of DNA at physiologically relevant concentrations. We introduce selective functionalization and localized electric field concentration to enhance aDSRR sensitivity and specificity. The sensor characteristics are demonstrated using the human tumor marker MIA in cDNA samples produced from total RNA without PCR-amplification. Measurements of DNA samples with concentrations as low as 1.55 × 10-12 mol/l are presented.

Discrimination of Nosiheptide Sources with Plasmonic Filters.

Bacteria identification plays a vital role in the field of clinical diagnosis, food industry, and environmental monitoring, which is in great demand of point of care detection methods. In this paper, in order to discriminate the source of nosiheptide product, a plasmonic filter was fabricated to filtrate, capture and identify Streptomycete spores with Surface enhanced Raman Scattering (SERS). Since the plasmonic filter was derived from self-assembled photonic crystal coated with silver, the plasmonic "hot spots" on the filter surface was distributed evenly in a fare good density and the SERS enhancement factor was 7.49 × 107. With this filter, a stain- and PCR-free detection was realized with only 5 μL sample solution and 5 min in a manner of "filtration and measure". Comparison to traditional Gram stain method and silver-plated nylon filter membrane, the plasmonic filter showed good sensitivity and efficiency in the discrimination of nosiheptide prepared with chemical and biological methods. It is anticipated that this simple SERS detection method with plasmonic filter has promising potentials in food safety, environmental, or clinical applications.

Demonstration of a tailorable and PCR-free detection of Enterococcus DNA isolated from soil samples

Bacteria detection is crucial for water and environmental monitoring.

Nanostructured CMOS Wireless Ultra-Wideband Label-Free PCR-Free DNA Analysis SoC

A fully integrated 54-channel wireless fast-scan cyclic voltammetry DNA analysis SoC is presented. The microsystem includes 546 3D nanostructured and 54 2D gold DNA sensing microelectrodes as well as 54 pH sensors. Each channel consists of a chopper-stabilized current conveyer with dynamic element matching. It is utilized as the amperometric readout circuit with a linear resolution from 8.6 pA to 350 nA. The on-chip programmable waveform generator provides a wide range of user-controlled rate and amplitude parameters with a maximum scan range of 1.2 V, and scan rate ranging between 0.1 mV/sec to 300 V/sec. A digital ultra-wideband transmitter based on a delay line architecture provides wireless data communication with data rates of up to 50 Mb/sec while consuming 400 µW. The 3 mm × 3 mm prototype fabricated in a 0.13 µm standard CMOS technology has been validated in prostate cancer synthetic DNA detection with 10 aM label-free PCR-free detection limit. Each channel occupies an area of only 0.06 mm 2 and consumes 42 µW of power from a 1.2 V supply.

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Functionalized gold nanoparticles for ultrasensitive DNA detection

A major challenge in the area of DNA detection is the development of rapid methods that do not require polymerase chain reaction (PCR) amplification of the genetic sample. The PCR amplification step increases the cost of the assay, the complexity of the detection, and the quantity of DNA required for the assay. In this context, methods that are able to perform DNA analyses with ultrasensitivity have recently been investigated with the aim of developing new PCR-free detection protocols. Functionalized gold nanoparticles have played a central role in the development of such methods. Here, possibilities offered by functionalized gold nanoparticle in the ultrasensitive detection of DNA are discussed. The different functionalization protocols available for gold nanoparticles and the principal DNA detection methods that are able to detect DNA at the femtomolar to attomolar level are presented.

A highly sensitive and specific biosensor for ligation- and PCR-free detection of MicroRNAs

A highly sensitive microRNA (miRNA) biosensor for both ligation- and PCR-free detection of miRNAs is described in this work. The biosensor was made of a monolayer of long oligonucleotide capture probes (CPs) comprising of miRNA capturing segments at the top (3′ termini) and detection probe capturing segments at the bottom (5′ termini). Following hybridization with a target miRNA, a cocktail of Surveyor ® and exonuclease I in pH 7.4 phosphate buffered saline was applied to the biosensor. Unhybridized CP strands and mismatched miRNA/CP duplexes were digested while complementarily hybridized ones remained intact. Thereafter, electrochemically activated glucose oxidase-tagged peptide nucleic acid detection probes (GOx–DP) were hybridized to the bottom segments of the CPs on the biosensor. The number of GOx molecules found at the biosensor surface coincides with the number of miRNA strands hybridized and therefore correlates directly to the concentration of the target miRNA. A detection limit of 10 fM and a linear current–concentration relationship up to 10 pM were attained under optimized conditions. The biosensor effectively eliminated the needs for chemical/biological ligation and PCR. It was showed that there is little cross-hybridization among closely related miRNA family members even at single-base-mismatched levels. Successful attempts were made in applying the biosensor to the detection of miRNAs in total RNA extracted from cell lines.

PCR-free detection of genetically modified organisms using magnetic capture technology and fluorescence cross-correlation spectroscopy.

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.

Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device

The labelfree detection of nucleic acid sequences is one of the modern attempts to develop quick, cheap and miniaturised hand-held devices for the future genetic testing in biotechnology and medical diagnostics. We present an approach to detect the hybridisation of DNA sequences using electrolyte-oxide-semiconductor field-effect transistors (EOSFETs) with micrometer dimensions. These semiconductor devices are sensitive to electrical charge variations that occur at the surface/electrolyte interface, i.e. upon hybridisation of oligonucleotides with complementary single-stranded (ss) oligonucleotides, which are immobilised on the oxide surface of the transistor gate. This method allows direct, time-resolved and in situ detection of specific nucleic acid binding events without any labelling. We focus on the detection mechanism of our sensors by using oppositely charged polyelectrolytes (PAH and PSS) subsequently attached to the transistor structures. Our results indicate that the sensor output is charge sensitive and distance dependent from the gate surface, which pinpoints the need for very defined surface chemistry at the device surface. The hybridisation of natural 19 base-pair sequences has been successfully detected with the sensors. In combination with nano-transistors a PCR free detection system might be feasible in future.