Additional Amplification Steps Research Articles

Functionalized Terpolymer-Brush-Based Biointerface with Improved Antifouling Properties for Ultra-Sensitive Direct Detection of Virus in Crude Clinical Samples.

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.

Electrochemistry-Assisted Surface Plasmon Resonance Biosensor for Detection of CA 15-3.

In this work, we describe an innovative methodology based on combined surface plasmon resonance (SPR) and electrochemical responses (eSPR) in the same immunoassay for screening CA 15-3 cancer biomarker with high sensitivity (and selectivity), in a very simple, label-free, accurate, and fully automated manner. Detection was achieved by performing two simple steps. In the first step, direct SPR was used to monitor CA 15-3 interaction with surface immobilized antibody. Two linear response ranges were obtained and the detection limit achieved is poor (LOD of 21 U mL-1). However, in the second detection step, electrochemical measurements at the SPR gold surface were performed to measure the decrease of redox probe peak current upon antigen-antibody interaction, providing a suitable amplification strategy to lower detection levels of CA 15-3 (LOD of 0.0998 U mL-1), without the need of additional complex and/or expensive amplification steps to enhance the sensitivity. Moreover, selectivity studies were performed against other common cancer biomarkers and the results showed that the eSPR immunosensor is selective for the CA 15-3 protein. Finally, the clinical applicability of the developed eSPR biosensing methodology was successfully applied to detect CA 15-3 in human serum samples at clinically relevant levels due to the high sensitivity of electrochemical readout. The same concept may be further extended to other proteins of interest.

Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction.

Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.

Highly portable quantitative screening test for prostate-specific antigen at point of care.

Prostate cancer (PCa) is the second most diagnosed cancer among men. Targeted PCa screening may decrease PCa-specific mortality. Prostate-specific antigen (PSA) is the most reliable and widely accepted tumor biomarker for screening and monitoring PCa status. However, in many settings, quantification of serum PSA requires access to centralized laboratories. In this study, we describe a proof-of-concept rapid test combined with a highly portable Cube™ reader for quantification of total PSA from a drop of serum within 20 min. We demonstrated the application of gold nanoshells as a label for lateral flow assay with significant increase in the measured colorimetric signal intensity to achieve five times lower detection limit when compared to the traditionally used 40 nm gold nanosphere labels, without a need for any additional signal amplification steps. We first optimized and evaluated the performance of the assay with commercially available total PSA calibrators. For initial validation with commercially available ACCESS Hybritech PSA calibrator, a detection range of 0.5–150 ng/mL was achieved. We compared the performance of our total PSA test with IMMULITE analyzer for quantification of total PSA in archived human serum samples. On preliminary testing with archived serums samples and comparison with IMMULITE total PSA assay, a correlation of 0.95 (p < .0001) was observed. The highly portable quantitative screening test for PSA described in this study has the potential to make PCa screening more accessible where diagnostic labs and automated immunoassay systems are not available, to reduce therapeutic turnaround time, to streamline clinical care, and to direct patient care for both initial screening and for post-treatment monitoring of patients.

Advancements in DNA-assisted Immunosensors

Several types of biosensors have been developed to detect a wide variety of human diseases. Immunosensors are classified as the most representative of all biosensors. They are based on antibodies that selectively recognize specific analytes and have high specificity and sensitivity. However, there are limitations to the types of substances that can be detected, and it is sometimes difficult to achieve sufficient sensitivity without additional amplification steps. To overcome these problems, novel immunosensors are being developed that combine DNA-based high signal amplification systems. These technologies ameliorate the low sensitivity of existing immunosensors by using DNA probes that can bind directly to targets as bioreceptors or act as signal amplifiers. In this review, we will discuss immunodetection methods that use DNA-based technologies on laboratory-scale and advanced point-of-care testing (POCT) that employ these technologies for high performance analyses.

Label-free sensitive detection of MUC1 using a liquid crystal based-system

ABSTRACT This study reports the development of a novel label-free aptasensor which uses the orientation properties of nematic liquid crystals (LCs) to detect mucin 1 (MUC1) is developed. The sensor is made of immobilised amine-functionalised aptamers on the surface, with LCs serving as response elements. When MUC1 interacts with the immobilised aptamers, it forms a complex that disrupts the LC orientation, taking them from homeotropic to random alignment. This change in the orientation of LCs is easily observed as a shift in the optical LC image from dark to bright using a polarised light microscope. This sensing system is highly sensitive (detection limit of 5 fg mL−1) and exhibits good specificity for detection of MUC1 without labelling or an additional amplification step.

Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

Titanium dioxide nanoparticle modified plasmonic interface for enhanced refractometric and biomolecular sensing.

A surface plasmon resonance (SPR) biosensor, which contains an overlayer of titanium dioxide nanoparticles (TDNPs) to modify the plasmonic interface, has been developed and investigated. Owing to its large surface area and high refractive index, the TDNP overlayer significantly enhances the probing electric field intensity and detection sensitivity. This sensitivity is related to the TDNP overlayer thickness, which can be engineered by changing the TiO2-ethanol dispersion's spin-coating concentration. The highest refractive index sensitivity for ethylene glycol measurement is 2567.3 nm/RIU, which is 38% higher than that of a conventional SPR sensor with an uncoated gold film. The proposed TDNP-SPR sensor also exhibits a 1.59-fold sensitivity enhancement in fetal bovine serum detection. Moreover, the proposed interface modification approach that is applied without additional biochemical amplification steps is chemical-free and contamination-free; therefore this TDNP-SPR sensor could be integrated into a sensitive, cost-effective, and biocompatible platform for rapid and label-free biochemical detection.

Plasmonic Interface Modified with Graphene Oxide Sheets Overlayer for Sensitivity Enhancement.

A novel strategy to modify the plasmonic interface by spin-coating an overlayer of graphene oxide sheets (GOSs) on top of the surface plasmon resonance (SPR) sensor is proposed and demonstrated. Thanks to the excellent electrical conductivity, large surface area, and high-refractive index of the GOSs layer, the GOSs-modified SPR (GOSs-SPR) sensor achieves an improved sensitivity in the detection of bulky refractive index solutions and bovine serum albumin (BSA) solutions.The maximum sensitivity of 2715.1 nm/RIU achieved by three spin-coatings shows an enhancement of 20.2% than the case without the modification of the GOSs overlayer. Benefiting from the large surface area and abundant surface functional groups, the GOSs-SPR sensor has a greater sensitivity enhancement (up to 39.35%) in the detection of the BSA molecules. Most importantly, we have firstly experimentally demonstrated that the GOSs overlayer with thickness over hundreds nanometers can still lead to a great enhancement of sensitivity of SPR sensors.Additionally, the proposed modification method for the plasmonic interface is a simple and effective strategy to boost the sensitivity in a chemical-free and environment-friendly manner, without additional chemical or biological amplification steps. These unique features make the proposed GOSs-SPR biosensor a low-cost and biocompatible platform in the fields of biochemical sensing, drug screening, and environmental monitoring.

Halloysite Nanotube-Modified Plasmonic Interface for Highly Sensitive Refractive Index Sensing.

We propose and demonstrate a novel strategy to modify the plasmonic interface by using a thin layer of halloysite nanotubes (HNTs). The modified surface plasmon resonance (SPR) sensor achieves a greatly improved sensitivity because the large surface area and high refractive index of the HNTs layer significantly increase the probing electric field intensity and hence the measurement sensitivity. More significantly, the thickness of the HNTs layer can be tailored by spraying different concentrations of HNTs ethanol suspension. The proposed sensors show significant superiority in terms of the highest sensitivity (10431 nm/RIU) and the enhancement fold (5.6-folds) over those reported previously. Additionally, the proposed approach is a chemical-free and environment-friendly modification method for the sensor interface, without additional chemical or biological amplification steps (no toxic solvents are used). These unique features make the proposed HNTs-SPR biosensor a simple, biocompatible, and low-cost platform for the trace-level detection of biochemical species in a rapid, sensitive, and nondestructive manner.

DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot.

Aims: Archived newborn bloodspots are valuable sample collections for genetic and epigenetic disease research. However, they have often been stored for long periods of time, under less than ideal circumstances, and nucleic acid yields can be low, particularly when samples become limiting. We wished to determine whether the quantity and quality of genomic DNA (gDNA) isolated from a single, surgical bloodspot punch (2 mm dia.) was adequate for accurate and reliable DNA methylome profiling on the Illumina HumanMethylation450 array.Methods: A total of 25–750 ng of archived bloodspot or Jurkat cell gDNA were bisulfite converted and analyzed on the array without any additional DNA amplification steps. Methylation profiles were assessed for call rate, call confidence (detection p-value), and reproducibility.Results: Using 25 ng gDNA from either Jurkat cells or dried bloodspots, array-wide call rates (∼99.9%) and detection p-values (99.9% with p < 5 × 10−6) were excellent. There was good agreement between methylation profiles generated from 25 ng gDNA and those generated from 750 ng (ρ > 0.98), although a fraction of CpG sites (2–8% depending on experiment) exhibited quantitative differences. Genome-wide methylation levels were strikingly reproducible from 25 ng DNA in both replicate and interindividual samples (ρ > 0.98).Conclusions: Twenty-five nanograms of gDNA, isolated from a single, surgical punch (2 mm dia.) of an archived newborn bloodspot, generate a genome-wide methylation profile on the Illumina HumanMethylation450 array that is robust, reproducible, and suitable for differential methylation studies.

Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling

Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2.) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling.

High Current (1225A) Optical Triggering of 18-kV 4H-SiC Thyristor in Purely Inductive Load Circuit

Optical switch-on of a very high voltage (18-kV class) 4H-SiC thyristor with an amplification step (pilot thyristor) to the current Imax = 1225A has been demonstrated using a purely inductive load. The results obtained show that a further switch-on current increase can only be achieved by introducing additional amplification steps in the pilot thyristor structure.

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries.

Detection of long-term influence of prenatal temperature stimulation on hypothalamic type-II iodothyronine deiodinase in juvenile female broiler chickens using a novel immunohistochemical amplification protocol

It has been clearly shown that early environmental stimulation may have long-lasting influence on body functions. Because of the strong relationship between thermoregulation and other homeostatic linked physiological parameters, perinatal thermal manipulation will also have an impact on other body functions like reproduction. As a maturation stimulant for later reproductive performance, hypothalamic type-2 iodothyronine deiodinase (Dio2) expression was investigated in 35day old immature female broilers with and without embryonic temperature stimulation. For the first time, human-specific Dio2 primary antibodies combined with additional amplification enabled the immunohistochemical detection of hypothalamic Dio2 protein in birds. The novel protocol includes an additional amplification step involving swine-anti-rabbit/mouse/goat antibodies against both goat anti-Dio2 primary and rabbit anti-goat biotinylated secondary commercial antibodies in the standard diaminobenzidine protocol. However, significant Dio2 expression was exclusively found in perinatally short-term temperature stimulated hens. Caudal but not rostral hypothalamic slices revealed that elevating incubation temperature by 1°C for 2h daily, from day 18 of embryonic development until hatching, induced a statistical significant expression of Dio2 within the subcommisural organ and the median eminence. This ample expression of Dio2 protein within caudal but not rostral hypothalamic slices of embryonic temperature stimulated chickens, leads to the assumption of a novel physiological prospective for embryonic thermal manipulation involving the suppression of thyroid hormone and the boosting of hypothalamic Dio2-induced FSH secretion to considerably advance the age of photoinduced egg production. It could be also of practicable relevance for broiler breeder females, and needs further investigations.

Optical triggering of 4H-SiC thyristors (18 kV class) to high currents in purely inductive load circuit

Optical switch-on of a very high voltage (18 kV class) 4H-SiC thyristor with an amplification step (pilot thyristor) to the current Imax = 1225 A is demonstrated using a purely inductive load and a calibrated air transformer. Increasing the inductance of the transformer primary winding slows down the turn on process. However, the inductance has little effect during the initial stage of the switch-on process when the voltage drop on the thyristor and its internal resistance is high. The results show that a further switch-on current increase can be only achieved by introducing additional amplification steps in the pilot thyristor.

Sensitivity and kinetic analysis of graphene oxide-based surface plasmon resonance biosensors

In this study, we demonstrate the fabrication of graphene oxide sheet (GOS)-based surface plasmon resonance (SPR) sensors for protein immobilization detection. GOS was used as the medium owing to its high covalent binding affinity for proteins. We were able to monitor the binding phenomenon in real time using the SPR-based technique. Thus, this GOS sensor can detect bovine albumin serum (BSA) directly in GOS films and exhibited up to a 4.3-fold higher angle shift than that of the conventional Au/Cr-based sensor in 10ng/ml of BSA. The results showed that the SPR biosensor formed using a GOS solution of 2mg/ml has optimal sensitive and had a BSA detection limit as low as 100pg/ml. The sensitivity of the slope ratio of the GOS vs. conventional Au substrate is 2.4:1. Moreover, GOS is a label-free detection without additional amplification steps to enhance the sensitivity. In addition, the average value of the affinity binding constant KA for the GOS-based sensor with respect to BSA was 80.8×106M−1. These results suggest that GOS-based SPR biosensors have potential for use in high-sensitivity protein immobilization detection.

Optical Triggering of High Current (1300 A), High-Voltage (12 kV) 4H-SiC Thyristor

A 12 kV class 4H-SiC thyristor with a pilot thyristor (an amplification step) has been triggered to a current Imax = 1310 A in a mixed resistive-inductive load circuit. In order to further increase the Imax, the homogeneity of the initially turned-on region should be improved and/or additional amplification steps introduced

High current (1300 A) optical triggering of a 12 kV 4H-SiC thyristor

Optical triggering of a high-voltage (12 kV class) 4H-SiC thyristor to a current Imax = 1300 A is demonstrated. An amplification step (pilot thyristor) and a mixed resistive-inductive load are used to reduce the current ramp dI/dt. The results obtained show that a decrease in the current density at the end of the first, fastest phase of the switch-on process can be achieved either by improving the homogeneity of the initially turned-on region or by introducing additional amplification steps.

Ultrasensitive and facile electrochemical deoxyribonucleic acid biosensor based on the conformational change of the recognition interface

An ultrasensitive electrochemical impedance spectroscopic deoxyribonucleic acid biosensor has been developed based on the conformational change of the deoxyribonucleic acid recognition interface with lodging probes. Pairing process leads to desorption of deoxyribonucleic acid bases from the gold surface, leading to a significant change of the interfacial conformation and the charge transfer resistance. Remarkably low detection limits down to 40fM are thus obtained without any additional amplification step.