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Scientific Reports Jul 2021In the present article, we developed a highly sensitive label-free electrochemical immunosensor based on NiFe-layered double hydroxides (LDH)/reduced graphene oxide...
In the present article, we developed a highly sensitive label-free electrochemical immunosensor based on NiFe-layered double hydroxides (LDH)/reduced graphene oxide (rGO)/gold nanoparticles modified glassy carbon electrode for the determination of receptor tyrosine kinase-like orphan receptor (ROR)-1. In this electrochemical immunoassay platform, NiFe-LDH/rGO was used due to great electron mobility, high specific surface area and flexible structures, while Au nanoparticles were prepared and coated on the modified electrodes to improve the detection sensitivity and ROR1 antibody immobilizing (ROR1Ab). The modification procedure was approved by using cyclic voltammetry and differential pulse voltammetry based on the response of peak current to the step by step modifications. Under optimum conditions, the experimental results showed that the immunosensor revealed a sensitive response to ROR1 in the range of 0.01-1 pg mL, and with a lower limit of quantification of 10 attogram/mL (10 ag mL). Furthermore, the designed immunosensor was applied for the analysis of ROR1 in several serum samples of chronic lymphocytic leukemia suffering patients with acceptable results, and it also exhibited good selectivity, reproducibility and stability.
Topics: Biomarkers; Biosensing Techniques; Electrochemical Techniques; Female; Ferric Compounds; Gold; Graphite; Humans; Hydroxides; Immunoassay; Leukemia, Lymphocytic, Chronic, B-Cell; Limit of Detection; Male; Metal Nanoparticles; Nanocomposites; Nickel; Receptor Tyrosine Kinase-like Orphan Receptors; Reproducibility of Results
PubMed: 34290319
DOI: 10.1038/s41598-021-94380-5 -
Scientific Reports Apr 2017Using doubly-clamped silicon (Si) microbeam resonators, we demonstrate sub-attogram per Hertz (ag/Hz) mass sensitivity, which is extremely high sensitivity achieved by...
Using doubly-clamped silicon (Si) microbeam resonators, we demonstrate sub-attogram per Hertz (ag/Hz) mass sensitivity, which is extremely high sensitivity achieved by micro-scale MEMS mass sensors. We also characterize unusual buckling phenomena of the resonators. The thin-film based resonator is composed of a Si microbeam surrounded by silicon nitride (SiN) anchors, which significantly improve performance by providing fixation on the microbeam and stabilizing oscillating motion. Here, we introduce two fabrication techniques to further improve the mass sensitivity. First, we minimize surface stress by depositing a sacrificial SiN layer, which prevents damage on the Si microbeam. Second, we modify anchor structure to find optimal design that allows the microbeam to oscillate in quasi-one dimensional mode while achieving high quality factor. Mass loading is conducted by depositing Au/Ti thin films on the local area of the microbeam surface. Using sequential mass loading, we test effects of changing beam dimensions, position of mass loading, and distribution of a metal film on the mass sensitivity. In addition, we demonstrate that microbeams suffer local micro-buckling and global buckling by excessive mass loading, which are induced by two different mechanisms. We also find that the critical buckling length is increased by additional support from the anchors.
PubMed: 28429793
DOI: 10.1038/srep46660 -
Angewandte Chemie (International Ed. in... Nov 2017Current trends in nanoengineering are bringing along new structures of diverse chemical compositions that need to be meticulously defined in order to ensure their...
Current trends in nanoengineering are bringing along new structures of diverse chemical compositions that need to be meticulously defined in order to ensure their correct operation. Few methods can provide the sensitivity required to carry out measurements on individual nano-objects without tedious sample pre-treatment or data analysis. In the present study, we introduce a pathway for the elemental identification of single nanoparticles (NPs) that avoids suspension in liquid media by means of optical trapping and laser-induced plasma spectroscopy. We demonstrate spectroscopic detection and identification of individual 25(±3.7) to 70(±10.5) nm in diameter Cu NPs stably trapped in air featuring masses down to 73±35 attograms. We found an increase in the absolute number of photons produced as size of the particles decreased; pointing towards a more efficient excitation of ensembles of only ca. 7×10 Cu atoms in the onset plasma.
PubMed: 28877398
DOI: 10.1002/anie.201708870 -
Frontiers in Cellular and Infection... 2023Periprosthetic joint infection (PJI) can be diagnosed to characterize the microorganisms constituting a biofilm, which is an essential procedure for proper treatment....
Periprosthetic joint infection (PJI) can be diagnosed to characterize the microorganisms constituting a biofilm, which is an essential procedure for proper treatment. The gold standard method for detecting and identifying the causative microorganism is culture of microorganisms from patients-derived sample.; however, this method takes a long time and has low sensitivity. To compensate for these limitations, identification methods based on real-time PCR (RT-PCR) have been widely used. However, RT-PCR also has limitations, including low sensitivity and the requirement of a standard curve for quantification. Therefore, to prevent significant proliferation of pathogenic bacteria, it is important to detect a limited number of infectious bacteria during early stages of PJI. In the present study, we developed droplet digital PCR-based detection of bacterial pathogens in PJI. And we evaluated the analytical performance of the assay using a model plasmid, based on the 16S ribosomal DNA sequence of target bacteria commonly found in PJI. We also prepared genomic DNA extracted from , , and to test whether ddPCR provides better sensitivity and quantification of the target sequences. ddPCR detected 400 attograms of target DNA, which was more than 10 times less than that detected by real-time PCR using synthesized plasmid. In addition, ddPCR detected target regions from genomic DNA of 50 femtograms for , 70 femtograms for , and 90 femtograms for . The results indicate that ddPCR has the potential to decrease the microbial detection limit and provide precise detection, signifying its effectiveness for early PJI.
Topics: Humans; Escherichia coli; Staphylococcus aureus; Plasmids; Bacteria; Arthritis, Infectious; Real-Time Polymerase Chain Reaction; DNA, Ribosomal
PubMed: 38029245
DOI: 10.3389/fcimb.2023.1301446 -
Talanta May 2018Interferometric imaging biosensors are powerful and convenient tools for confirming the existence of DNA monolayer films on silicon microarray platforms. However, their...
Interferometric imaging biosensors are powerful and convenient tools for confirming the existence of DNA monolayer films on silicon microarray platforms. However, their accuracy and sensitivity need further improvement because DNA molecules contribute to an inconspicuous interferometric signal both in thickness and size. Such weaknesses result in poor performance of these biosensors for low DNA content analyses and point mutation tests. In this paper, an interferometric imaging biosensor with weighted spectrum analysis is presented to confirm DNA monolayer films. The interferometric signal of DNA molecules can be extracted and then quantitative detection results for DNA microarrays can be reconstructed. With the proposed strategy, the relative error of thickness detection was reduced from 88.94% to merely 4.15%. The mass sensitivity per unit area of the proposed biosensor reached 20 attograms (ag). Therefore, the sample consumption per unit area of the target DNA content was only 62.5 zeptomoles (zm), with the volume of 0.25 picolitres (pL). Compared with the fluorescence resonance energy transfer (FRET), the measurement veracity of the interferometric imaging biosensor with weighted spectrum analysis is free to the changes in spotting concentration and DNA length. The detection range was more than 1µm. Moreover, single nucleotide mismatch could be pointed out combined with specific DNA ligation. A mutation experiment for lung cancer detection proved the high selectivity and accurate analysis capability of the presented biosensor.
Topics: Algorithms; Biosensing Techniques; DNA; DNA Mutational Analysis; Fluorescence Resonance Energy Transfer; Humans; Interferometry; Lung Neoplasms; Point Mutation; Reproducibility of Results
PubMed: 29426505
DOI: 10.1016/j.talanta.2017.12.066 -
Analytica Chimica Acta Mar 2015A pyrene-based small molecular weight probe, exhibiting aggregation enhanced excimer emission has been synthesized. The crystalline emissive form detects...
A pyrene-based small molecular weight probe, exhibiting aggregation enhanced excimer emission has been synthesized. The crystalline emissive form detects 2,4,6-trinitrophenol (picric acid) at parts-per-billion concentration in solution and as low as 0.46 attogram in direct contact mode, operating predominantly in a static quenching mechanism, proposed on the basis of steady state and life-time fluorescence measurements.
Topics: Fluorescent Dyes; Picrates; Pyrenes; Software; Spectrometry, Fluorescence
PubMed: 25732427
DOI: 10.1016/j.aca.2015.01.029 -
Environmental Science & Technology Feb 2018Quantifying metal and nanoparticle (NP) biouptake and distribution on an individual cellular basis has previously been impossible, given available techniques which...
Quantifying metal and nanoparticle (NP) biouptake and distribution on an individual cellular basis has previously been impossible, given available techniques which provide qualitative data that are laborious to acquire and prone to artifacts. Quantifying metal and metal NP uptake and loss processes in environmental organisms will lead to mechanistic understanding of biouptake and improved understanding of potential hazards and risks of metals and NPs. In this work, we present a new technique, single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), which allows quantification of metal concentrations on an individual cell basis down to the attogram (ag) per cell level. We present data validating the novel method, along with the mass of metal per cell. Finally, we use SC-ICP-MS, with ancillary cell counting methods, to quantify the biouptake and strong sorption and distribution of both dissolved Au and Au NPs in a freshwater alga (Cyptomonas ovate). The data suggests differences between dissolved and NP uptake and loss. In the case of NPs, there was a dose and time dependent uptake, but individual cellular variations; at the highest realistic exposure conditions used in this study up to 40-50% of cells contained NPs, while 50-60% of cells did not.
Topics: Fresh Water; Gold; Mass Spectrometry; Metal Nanoparticles; Particle Size
PubMed: 29400052
DOI: 10.1021/acs.est.7b04968 -
Microbiome Apr 2019Metagenomic next-generation sequencing (mNGS) experiments involving small amounts of nucleic acid input are highly susceptible to erroneous conclusions resulting from...
Metagenomic next-generation sequencing (mNGS) experiments involving small amounts of nucleic acid input are highly susceptible to erroneous conclusions resulting from unintentional sequencing of occult contaminants, especially those derived from molecular biology reagents. Recent work suggests that, for any given microbe detected by mNGS, an inverse linear relationship between microbial sequencing reads and sample mass implicates that microbe as a contaminant. By associating sequencing read output with the mass of a spike-in control, we demonstrate that contaminant nucleic acid can be quantified in order to identify the mass contributions of each constituent. In an experiment using a high-resolution (n = 96) dilution series of HeLa RNA spanning 3-logs of RNA mass input, we identified a complex set of contaminants totaling 9.1 ± 2.0 attograms. Given the competition between contamination and the true microbiome in ultra-low biomass samples such as respiratory fluid, quantification of the contamination within a given batch of biological samples can be used to determine a minimum mass input below which sequencing results may be distorted. Rather than completely censoring contaminant taxa from downstream analyses, we propose here a statistical approach that allows separation of the true microbial components from the actual contribution due to contamination. We demonstrate this approach using a batch of n = 97 human serum samples and note that despite E. coli contamination throughout the dataset, we are able to identify a patient sample with significantly more E. coli than expected from contamination alone. Importantly, our method assumes no prior understanding of possible contaminants, does not rely on any prior collection of environmental or reagent-only sequencing samples, and does not censor potentially clinically relevant taxa, thus making it a generalized approach to any kind of metagenomic sequencing, for any purpose, clinical or otherwise.
Topics: DNA, Bacterial; Escherichia coli; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Sequence Analysis, DNA
PubMed: 30992055
DOI: 10.1186/s40168-019-0678-6 -
ACS Sensors Apr 2020As the use of nanoparticles is expanding in many industrial sectors, pharmaceuticals, cosmetics among others, flow-through characterization techniques are often required...
As the use of nanoparticles is expanding in many industrial sectors, pharmaceuticals, cosmetics among others, flow-through characterization techniques are often required for in-line metrology. Among the parameters of interest, the concentration and mass of nanoparticles can be informative for yield, aggregates formation or even compliance with regulation. The Suspended Nanochannel Resonator (SNR) can offer mass resolution down to the attogram scale precision in a flow-through format. However, since the readout has been based on the optical lever, operating more than a single resonator at a time has been challenging. Here we present a new architecture of SNR devices with piezoresistive sensors that allows simultaneous readout from multiple resonators. To enable this architecture, we push the limits of nanofabrication to create implanted piezoresistors of nanoscale thickness (∼100 nm) and implement an algorithm for designing SNRs with dimensions optimized for maintaining attogram scale precision. Using 8-in. processing technology, we fabricate parallel array SNR devices which contain ten resonators. While maintaining a precision similar to that of the optical lever, we demonstrate a throughput of 40 000 particles per hour-an order of magnitude improvement over a single device with an analogous flow rate. Finally, we show the capability of the SNR array device for measuring polydisperse solutions of gold particles ranging from 20 to 80 nm in diameter. We envision that SNR array devices will open up new possibilities for nanoscale metrology by measuring not only synthetic but also biological nanoparticles such as exosomes and viruses.
Topics: Gold; Microfluidic Analytical Techniques; Nanoparticles
PubMed: 32233476
DOI: 10.1021/acssensors.0c00394 -
Clinical Infectious Diseases : An... Apr 2020Treatment of subarachnoid neurocysticercosis (NCC) is complicated, and assays that can guide treatment are not widely available. The reproducibility and scalability of...
A Novel, Highly Sensitive Quantitative Polymerase Chain Reaction Assay for the Diagnosis of Subarachnoid and Ventricular Neurocysticercosis and for Assessing Responses to Treatment.
BACKGROUND
Treatment of subarachnoid neurocysticercosis (NCC) is complicated, and assays that can guide treatment are not widely available. The reproducibility and scalability of molecular-based biomarkers would be of great use.
METHODS
The Taenia solium genome was mined and primers and probes were designed to target repeats with the highest coverage; the most sensitive, specific, and efficient repeat (TsolR13) was selected for clinical testing. We tested 46 plasma samples and 36 cerebral spinal fluid (CSF) samples taken from patients with subarachnoid or ventricular disease using quantitative polymerase chain reaction (qPCR).
RESULTS
The analytic sensitivity of TsolR13 was 97.3% at 240 attograms (ag) of T. solium genomic DNA and 100% analytic specificity. The clinical sensitivity in detecting active subarachnoid or ventricular disease in symptomatic patients was 100% in CSF and 81.3% in plasma. The predictive ability to distinguish active from cured disease was better for CSF (94.4% of those cured had negative qPCR results) than for plasma (86.7% of those cured tested negative). Some subjects also had plasma DNA detectable intermittently for years after being cured. Overall, the test performance was equivalent to T. solium antigen detection.
CONCLUSIONS
A qPCR test for the detection of the highly repetitive Tsol13 sequence has been developed and shown to be highly sensitive and specific for NCC, but also useful as a test of cure in CSF and for the definitive diagnosis of NCC in plasma.
Topics: Animals; Antigens, Helminth; Enzyme-Linked Immunosorbent Assay; Humans; Neurocysticercosis; Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity; Taenia solium
PubMed: 31232448
DOI: 10.1093/cid/ciz541