Ultrasensitive Assay Research Articles

Development of a digital anti-Müllerian hormone immunoassay: ultrasensitive, accurate and practical strategy for reduced ovarian reserve monitoring and assessment

Anti-Müllerian hormone (AMH) is an ideal biomarker for the assessment of ovarian reserve. However, its application in determining ovarian reserve reduction is restricted due to the low sensitivity of existing AMH assays. Herein, a homebrew ultrasensitive digital AMH assay (UD-AMH) was established based on a single-molecule array (SiMoA, HD-X platform), and the analytical performance of UD-AMH was evaluated systematically. The limit of detection (LoD) and limit of quantitation (LoQ) of UD-AMH were 0.13 and 0.14 pg/mL, respectively, which is approximately 100-fold higher than that of the current reported general clinical AMH assay. A comparison study showed a high correlation, with r = 0.988 for the Beckman Access AMH assay and r = 0.945 for the Kangrun AMH assay. In addition, we found that the AMH concentrations of premature ovarian insufficiency (POI) patients were very low (2.59 (0.86, 31.79) pg/mL) and similar to those of perimenopausal women (2.37 (0.65, 35.88) pg/mL) but significantly higher than those of menopausal women (0.43 (0.28, 1.17) pg/mL). Furthermore, we observed that the AMH concentration of most hormone therapy (HT) treated POI patients decreased sharply, suggesting that the ovarian reserve of POI patients declines over time even under HT-treatment.

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay

BackgroundIntegrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens.MethodsRT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored.ResultsThe results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/μL within approximately 30–40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement.ConclusionsTogether, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

Ultrasensitive monitoring of SARS-CoV-2-specific antibody responses based on a digital approach reveals one week of IgG seroconversion

COVID-19 is still unfolding, while many people have been vaccinated. In comparison to nucleic acid testing (NAT), antibody-based immunoassays are faster and more convenient. However, its application has been hampered by its lower sensitivity and the existing fact that by traditional immunoassays, the measurable seroconversion time of pathogen-specific antibodies, such as IgM or IgG, lags far behind that of nucleic acids. Herein, by combining the single molecule array platform (Simoa), RBD, and a previously identified SARS-CoV-2 S2 protein derivatized 12-aa peptide (S2-78), we developed and optimized an ultrasensitive assay (UIM-COVID-19 assay). Sera collected from three sources were tested, i.e., convalescents, inactivated virus vaccine-immunized donors and wild-type authentic SARS-CoV-2-infected rhesus monkeys. The sensitivities of UIM-COVID-19 assays are 100–10,000 times higher than those of conventional flow cytometry, which is a relatively sensitive detection method at present. For the established UIM-COVID-19 assay using RBD as a probe, the IgG and IgM seroconversion times after vaccination were 7.5 and 8.6 days vs. 21.4 and 24 days for the flow cytometry assay, respectively. In addition, using S2-78 as a probe, the UIM-COVID-19 assay could differentiate COVID-19 patients (convalescents) from healthy people and patients with other diseases, with AUCs ranging from 0.85–0.95. In summary, the UIM-COVID-19 we developed here is a promising ultrasensitive biodetection strategy that has the potential to be applied for both immunological studies and diagnostics.

Ultrasensitive assay of HER1, HER2, and heregulin-α in whole blood

Microsensors based on the physical immobilization of oleamides (N-(pyridine-3-yl-methyl) oleamide, and N-(2-mercapto-1H-benzo[d]imidazole-5-yl) oleamide) on paste containing nanographene decorated with spheroidal Cu were proposed for the molecular recognition and ultrasensitive simultaneous determination of HER1, HER2, and heregulin-α in whole blood. While for the assay of HER1 and HER2 there were not significant differences in the sensitivities recorded when N-(pyridine-3-yl-methyl) oleamide, and N-(2-mercapto-1H-benzo[d]imidazole-5-yl) oleamide were used to modify the nanographene paste, for the assay of heregulin-α, a higher sensitivity was recorded when N-(2-mercapto-1H-benzo[d]imidazole-5-yl) oleamide was used to modify the paste. Wide working concentration ranges were recorded for HER1 (between 10−13 and 10−8 mol L − 1), HER2 (between 10−12–10−10 mol L − 1), and heregulin-α (10−13–10−11mol L − 1). The student t-test showed that there is no significant difference between the results obtained by utilizing the two microsensors for screening tests of whole blood, at 99% confidence level.

Ultra-Sensitive and Semi-Quantitative Vertical Flow Assay for the Rapid Detection of Interleukin-6 in Inflammatory Diseases.

The inflammation biomarker Interleukin 6 (IL-6) exhibits a concentration of less than 7 pg/mL in healthy serum but increases 10–100-fold when inflammation occurs. Increased serum IL-6 has been reported in chronic diseases such as rheumatoid arthritis (RA), as well as in life-threatening acute illnesses such as sepsis and cytokine release syndrome (CRS). This work seeks to meet the demand for rapid detection of serum IL-6 both for rapid monitoring of chronic diseases and for triaging patients with acute illnesses. Following the optimization of several types of gold nanoparticles, membrane pore sizes, and buffer systems, an ultra-sensitive vertical flow assay (VFA) was engineered, allowing the detection of recombinant IL-6 in spiked buffer with a limit of detection (LoD) of 10 pg/mL and a reportable range of 10–10,000 pg/mL with a 15-min assay time. The detection of IL-6 in spiked pooled healthy serum exhibited an LoD of 3.2 pg/mL and a reportable range of 10–10,000 pg/mL. The VFA’s stability was demonstrated over 1-day, two-week, four-week, and six-week storage durations at room temperature. The inter-operator CV and intra-operator CV were determined to be 14.3% and 15.2%, respectively. Three reference zones, high, low, and blank, were introduced into the cartridge to facilitate on-site semi-quantitative measurements across a 6-point semi-quantitative range. Finally, the performance of the IL-6 VFA was validated using 20 RA and 20 healthy control (HC) clinical serum samples, using ELISA as the gold standard platform. The ultra-sensitive, rapid IL-6 VFA could potentially be used to triage patients for intensive care, treatment adjustments, or for monitoring disease activity in inflammatory conditions.

Immunoarray Measurements of Parathyroid Hormone-Related Peptides Combined with Other Biomarkers to Diagnose Aggressive Prostate Cancer.

Parathyroid hormone-related peptide (PTHrP) is related to bone metastasis and hypercalcemia in prostate and breast cancers and should be an excellent biomarker for aggressive forms of these cancers. Current clinical detection protocols for PTHrP are immunoradiometric assay and radioimmunoassay but are not sensitive enough to detect PTHrPs at early stages. We recently evaluated a prostate cancer biomarker panel, including serum monocyte differentiation antigen (CD-14), ETS-related gene protein, pigment epithelial-derived factor, and insulin-like growth factor-1, with promise for identifying aggressive prostate cancers. This panel predicted the need for patient biopsy better than PSA alone. In the present paper, we report an ultrasensitive microfluidic assay for PTHrPs and evaluate their diagnostic value and the value of including them with our prior biomarker panel to diagnose aggressive forms of prostate cancer. The immunoarray features screen-printed carbon sensor electrodes coated with 5 nm glutathione gold nanoparticles with capture antibodies attached. PTHrPs are bound to a secondary antibody attached to a polyhorseradish peroxidase label and delivered to the sensors to provide high sensitivity when activated by H2O2 and a mediator. We obtained an unprecedented 0.3 fg mL-1 limit of detection for PTHrP bioactive moieties PTHrP 1-173 and PTHrP 1-86. We also report the first study of PTHrPs in a large serum pool to identify aggressive malignancies. In assays of 130 human patient serum samples, PTHrP levels distinguished between aggressive and indolent prostate cancers with 83-91% clinical sensitivity and 78-96% specificity. Logistic regression identified the best predictive model as a combination of PTHrP 1-86 and vascular endothelial growth factor-D. PTHrP 1-173 alone also showed a high ability to differentiate aggressive and indolent cancers.

Carbon Nanopowder-Based Stochastic Sensor for Ultrasensitive Assay of CA 15-3, CEA and HER2 in Whole Blood.

Two microsensors obtained by the physical immobilization of 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP) and 5,10,15,20-tetrakis (pentafluorophenyl chloride)-21H,23H-iron (III) porphyrin (Fe(TPFPP)Cl) in carbon nanopowder decorated with gold nanoparticles (AuNp) were designed, characterized, validated and used for the molecular recognition and simultaneous ultrasensitive determination of CEA, CA15-3 and HER2 in whole blood. High sensitivities were recorded for both microsensors. Low limits of quantification were recorded for all biomarkers: CEA (12.8 pg mL−1 by using Fe(TPFPP)Cl/AuNp, and 190 fg mL−1 by using TPP/AuNp), CA 15-3 (100 fU mL−1 for both microsensors) and HER2 (3.9 fg mL−1 by using Fe(TPFPP)Cl/AuNp, and 35 fg mL−1 by using TPP/AuNp). A very good correlation between the results obtained using the proposed microsensors and ELISA, certified by the Student t-test, proves that the screening test can be used for ultrasensitive assays of the three biomarkers in whole blood.

Portable chemiluminescence optical fiber aptamer-based biosensors for analysis of multiple mycotoxins

Multiple mycotoxin contamination in foodstuffs causes significant threat to human health especially considering their synergistic effects. Herein, we present a novel chemiluminescence optical fiber aptamer-based biosensors for ultrasensitive on-site assay of multiplex mycotoxins in food samples. The optical fiber aptasensor is developed based on the specific recognition of single-stranded binding protein (SSB) and mycotoxin aptamers for the selective analysis. Optical fibers are utilized not only as the carrier to immobilize SSB to prepare the sensing region, but also as the transducer of the chemiluminescent emission to detect the sensor response. Multiple-channel fiber sensors are coupled together toward the detector and a compact and portable design of the chemiluminescence optical fiber aptasensor system is proposed, allowing four mycotoxins (AFB1, FB1, OTA or ZEN) to be simultaneously determined from one run without any tedious operation procedure. The optical fiber aptasensor system can perform sensitive analysis with a wide detection range from pg/mL to 105 pg/mL and low limit-of-detection in the range of 0.015 pg/mL-0.423 pg/mL for different target mycotoxins. The method is successfully applied for simultaneous and sensitive detection of multiple mycotoxins in infant cereals, showing its practical application in on-site analysis of real food samples.

380P Tumour naïve circulating tumour DNA (ctDNA) analysis in patients (pts) with resected colorectal cancer (CRC) in the phase III ASCOLT trial

Plasma ctDNA detects minimal residual disease (MRD) and is associated with disease recurrence in curatively treated CRC. In a randomised, double-blinded, phase III study (The ASpirin for Dukes C and high risk Dukes B COLorecTal cancer trial [ASCOLT], NCT00565708) evaluating the benefit of adjuvant aspirin, pts in the Australian and New Zealand cohort underwent prospective collection of plasma at baseline (within 90 days of completing adjuvant chemotherapy), 6 and 12 months, as well as surgical specimens. In a pilot cohort of 51 pts, we studied the utility of an ultra-sensitive ctDNA assay (SafeSEQ, Sysmex Inostics) for MRD detection. Cell-free DNA (cfDNA) analysis used the SafeSEQ CRC MRD assay (14 genes, AKT1, APC, BRAF, CTNNB1, ERBB3, FBXW7, KRAS, NRAS, PIK3CA, POLE, PPP2R1A, RNF43, SMAD4 and TP53). cfDNA results were correlated with recurrence data and tumour sequencing results using a 59-gene custom panel. All pts (26 female; median age 69; 16 high risk stage II, 35 III) received adjuvant 5-Fluorouracil based chemotherapy (32 with oxaliplatin, 4 had neoadjuvant chemoradiation for rectal cancer) and median follow-up was 60 months. 133 plasma samples were analysed (median 3.8 mL and DNA 71.0 genomic equivalents/ μL). Of the 10 pts with known recurrences (median time to recurrence 19 months), 6 had detectable cfDNA, including 2 of 3 pts without detectable tumour mutations (Table); median time from positive cfDNA to imaging detected recurrence was 28 weeks. Four pts with known recurrence did not have detectable cfDNA, including 1 who recurred 59 months after enrolment. Fourteen pts had plasma mutations without known recurrence. Matched peripheral blood mononuclear cell analyses are awaited.Table: 380PcfDNA mutationsMutant allele fraction (%, 1st sample if multiple)cfDNA detected at baselineAt 6mAt 12mConcordant mutation in tumourTime from positive cfDNA to recurrence on CT (w)Time from enrolment to recurrence (w)CEA elevated at CT recurrenceRecurrence site(s)BRAF V600ETP53 C275Y13.20.05Y- (Not done)-YN05YLiverSMAD4 R496C0.08NNYN4799YLiver, new primarynoneNNN257NPeritoneumKRAS G12VTP53 I195STP53 V272M0.040.160.04NYNYYYYYNYNN101101NLungKRAS G13DSMAD4 540PAPC R1399fs*9TP53 R342*8.42.42.56.5NNNNNNNNYYYYYYNN048NLung, nodalnoneNNN80NLung, nodalTP53 Y220C0.03-Y-N7198YNodal, localKRAS G12VTP53 R306*0.040.07NYYN-YN024NLungnoneNN-30NLungnoneNNN87NLung Open table in a new tab In 51 ASCOLT pts with high risk resected CRC following adjuvant chemotherapy, 6 of 10 with reported recurrences had detectable cfDNA using a tumour naïve SafeSEQ assay. Analyses in the larger cohort (n = 368) are underway.

Magnetic Nanotag-Based Colorimetric/SERS Dual-Readout Immunochromatography for Ultrasensitive Detection of Clenbuterol Hydrochloride and Ractopamine in Food Samples.

Direct and sensitive detection of multiple illegal additives in complex food samples is still a challenge in on-site detection. In this study, an ultrasensitive immunochromatographic assay (ICA) using magnetic Fe3O4@Au nanotags as a capture/detection difunctional tool was developed for the direct detection of β2-adrenoceptor agonists in real samples. The Fe3O4@Au tag is composed of a large magnetic core (~160 nm), a rough Au nanoshell, dense surface-modified Raman molecules, and antibodies, which cannot only effectively enrich targets from complex solutions to reduce the matrix effects of food samples and improve detection sensitivity, but also provide strong colorimetric/surface-enhanced Raman scattering (SERS) dual signals for ICA testing. The dual readout signals of the proposed ICA can meet the detection requirements in different environments. Specifically, the colorimetric signal allows for rapid visual detection of the analyte, and the SERS signal is used for the sensitive and quantitative detection modes. The proposed dual-signal ICA can achieve the simultaneous determination of two illegal additives, namely, clenbuterol hydrochloride and ractopamine. The detection limits for the two targets via colorimetric and SERS signals were down to ng mL−1 and pg mL−1 levels, respectively. Moreover, the proposed assay has demonstrated high accuracy and stability in real food samples.

Electrochemiluminescent (ECL) biosensor for Burkholderia pseudomallei based on cobalt-doped MOF decorated with gold nanoparticles and N-(4-aminobutyl)-N-(ethylisoluminol).

A multifunctional catalytic nanomaterial (Co-MOF@AuNP@ABEI) composed of cobalt-doped metal-organic frameworks (Co-MOF), gold nanoparticles (AuNP), and N-(4-aminobutyl)-N-(ethylisoluminol) (ABEI) is reported. Co-MOF@AuNP@ABEI exhibits high synergistic and zero-distance catalytic properties, which are beneficial to the improvement of the detection sensitivity of an electrochemiluminescent (ECL) biosensor. After coupling with the ECL system and 3D magnetic walking nanomachine amplification strategy, the Co-MOF@AuNP@ABEI can achieve an ultrasensitive ECL assay of Burkholderia pseudomallei with the limit of detection (LOD) of 60.3 aM, which is 2 and 4 orders of magnitude lower than individual ECL system without the nanomachine (4.97 fM) and individual walking nanomachine (340 fM), and superior to the pathogenic bacteria analyses in the previous report. Moreover, the LOD of the proposed ECL detection system for the determination of B. pseudomallei in serum sample was as low as 9.0CFUmL-1. The relative standard deviations (RSD) of ECL intensity for the detection of five B. pseudomallei-spiked serum samples were 4.02%, 0.84%, 0.84%, 1.55%, and 0.21%, respectively. The recoveries of the ECL biosensor for the detection of B. pseudomallei DNA-spiked serum samples were 93.63 ~ 107.83%. Therefore, this work demonstrated that the developed multifunctional catalytic nanomaterial with synergistic and zero-distance catalytic properties can be used as excellent ECL signal reporter to improve the detection sensitivity of ECL biosensor.

Highly selective and multicolor ultrasensitive assay of dipicolinic acid: The integration of terbium(III) and gold nanocluster

A novel multicolor fluorescent nano-probe based on the hybridization of Tb3+ ion with gold nanoclusters (Au NCs) was synthesized to monitor and on-site visual assay of 2,6-pyridinedicarboxylic acid (DPA), a biomarker of bacterial spores. DPA can replace the water molecule in the center of Tb3+ and strongly coordinate with Tb3+ based on the analyte-triggered antenna effect. Simultaneously, the red fluorescence of Au NCs is not influenced after addition of DPA and can be used as steady inside fluorescence reference channel to measure background noise. On this basis, the multicolor fluorescence nano-probe based on Tb3+-doped Au NCs for fast analysis of DPA was fabricated. The linear range of this method is 0 to 12.5 μM and the limit of detection is 3.4 nM, which is well below the quantity of DPA concentration of 60 μM released by the spore transmission dose of anthrax infection. The proposed multicolor fluorescence nano-probe was successfully detecting DPA in actual sample with good sensitivity and specificity. In addition, the visual paper-based nano-probe is designed to detect DPA by using the color scanning application of smart phone. This developed platform possesses abroad application prospects with advantages of effective, convenient carrying, simple operation, good selectivity and repeatability.

Plasma β-amyloid, tau, neurodegeneration biomarkers and inflammatory factors of probable Alzheimer's disease dementia in Chinese individuals.

BackgroundPlasma-derived β-amyloid, tau, and neurodegeneration (ATN) biomarkers can accurately diagnose Alzheimer’s disease (AD) and predict its progression. Few studies have investigated the relationship between plasma biomarkers and changes in plasma inflammatory markers in clinically diagnosed AD.MethodsSeventy-four participants were recruited, including 30 mild-to-moderate AD dementia patients and 44 normal controls (NC). All participants underwent neuropsychological testing and blood sampling for biomarker testing. AD was clinically diagnosed according to the National Institute on Aging-Alzheimer’s Association (NIA-AA) core criteria and required age-mismatched hippocampal atrophy. We performed Single Molecule Array (Simoa), an ultra-sensitive enzyme-linked immunosorbent assay (ELISA), to examine plasma ATN markers, including β-amyloid (Aβ) 40, Aβ42, p-tau181, total (t)-tau, neurofilament protein light chain (NfL), and inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8).ResultsThe level of the plasma Aβ42/Aβ40 ratio was significantly declined and the levels of the plasma p-tau181, NfL and TNF-α were significantly higher in the AD group than the NC group, but there was no significant difference in the levels of plasma t-tau, IL-1β, IL-6, and IL-8 between the AD and NC groups. The levels of plasma p-tau181, NfL, Aβ42/Aβ40 ratio, and TNF-α were all associated with impairments in multiple cognitive domains. Among them, the plasma Aβ42/Aβ40 ratio, and the p-tau181 and TNF-α levels were associated with impairments in global cognition, memory, and visuospatial abilities, but not with executive function, only plasma NfL level was associated with executive function. Plasma NfL showed higher diagnostic performance in AD than in NC individuals (AUC = 0.833). A combined diagnostic prediction model of plasma Aβ42/Aβ40 ratio, p-tau 181, and NfL had the highest value than each factor alone (AUC = 0.902),with a sensitivity and specificity of 0.867 and 0.886, respectively.ConclusionThe levels of plasma ATN biomarkers (Aβ42/Aβ40 ratio, p-tua181, and NfL) were significantly changed in clinically diagnosed AD patients and they all associated with different domains of cognitive impairment. Plasma ATN biomarkers better differentiate mild-to-moderate AD dementia from NC when they are incorporated into diagnostic models together rather than individually. Plasma ATN biomarkers have the potential to be a screening tool for AD. However, the expression of inflammatory factors in AD patients requires further research.

A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

CRISPR diagnostics are powerful tools for detecting nucleic acids but are generally not deployable for the detection of clinically important proteins. Here, we report an ultrasensitive CRISPR-based antibody detection (UCAD) assay that translates the detection of anti-SARS-CoV-2 antibodies into CRISPR-based nucleic acid detection in a homogeneous solution and is 10,000 times more sensitive than the classic immunoassays. Clinical validation using serum samples collected from the general population (n = 197), demonstrates that UCAD has 100% sensitivity and 98.5% specificity. With ultrahigh sensitivity, UCAD enables the quantitative analysis of serum anti-SARS-CoV-2 levels in vaccinated kidney transplant recipients who are shown to produce “undetectable” anti-SARS-CoV-2 using standard immunoassay. Because of the high sensitivity and simplicity, we anticipate that, upon further clinical validation against large cohorts of clinical samples, UCAD will find wide applications for clinical uses in both centralized laboratories and point-of-care settings.

Technical Validation and Clinical Implications of Ultrasensitive PCR Approaches for EGFR-Thr790Met Mutation Detection in Pretreatment FFPE Samples and in Liquid Biopsies from Non-Small Cell Lung Cancer Patients.

In pretreatment tumor samples of EGFR-mutated non-small cell lung cancer (NSCLC) patients, EGFR-Thr790Met mutation has been detected in a variable prevalence by different ultrasensitive assays with controversial prognostic value. Furthermore, its detection in liquid biopsy (LB) samples remains challenging, being hampered by the shortage of circulating tumor DNA (ctDNA). Here, we describe the technical validation and clinical implications of a real-time PCR with peptide nucleic acid (PNA-Clamp) and digital droplet PCR (ddPCR) for EGFR-Thr790Met detection in diagnosis FFPE samples and in LB. Limit of blank (LOB) and limit of detection (LOD) were established by analyzing negative and low variant allele frequency (VAF) FFPE and LB specimens. In a cohort of 78 FFPE samples, both techniques showed an overall agreement (OA) of 94.20%. EGFR-Thr790Met was detected in 26.47% of cases and was associated with better progression-free survival (PFS) (16.83 ± 7.76 vs. 11.47 ± 1.83 months; p = 0.047). In LB, ddPCR was implemented in routine diagnostics under UNE-EN ISO 15189:2013 accreditation, increasing the detection rate of 32.43% by conventional methods up to 45.95%. During follow-up, ddPCR detected EGFR-Thr790Met up to 7 months before radiological progression. Extensively validated ultrasensitive assays might decipher the utility of pretreatment EGFR-Thr790Met and improve its detection rate in LB studies, even anticipating radiological progression.

An ultrasensitive nanofiber-based assay for enzymatic hydrolysis and deep-sea microbial degradation of cellulose

SummarySubstrates for enzymatic reactions, such as cellulose and chitin, are often insoluble in water. The enzymatic degradation of these abundant organic polymers plays a dominant role in the global carbon cycle and has tremendous technological importance in the production of bio-based chemicals. In addition, biodegradation of plastics is gaining wide attention. However, despite the significance, assaying these degradation reactions remains technically challenging owing to the low reaction rate, because only the surface of the substrate is accessible to the enzymes. We developed a nanofiber-based assay for the enzymatic hydrolysis of cellulose. This assay facilitated the quantification of the enzymatic hydrolysis of <1 ng crystalline cellulose. Utilization of the assay for the functional screening of cellulolytic microorganisms revealed an unprecedented genetic diversity underlying the production of deep-sea cellulase. This study reiterates that interdisciplinary efforts, such as from nanotechnology to microbiology, are critical for solving sustainability challenges.

One-pot and rapid detection of SARS-CoV-2 viral particles in environment using SERS aptasensor based on a locking amplifier

With the frequent detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in dwellings and wastewater, the risk of transmission of environmental contaminants is of great concern. Fast, simple and sensitive sensors are essential for timely detecting infection and controlling transmission through environment fomites. Herein, we developed a Surface Enhanced Raman Scattering (SERS) aptasensor, which can realize ultrasensitive and rapid assay of SARS-CoV-2 viral particles. In this strategy, we designed a novel locking amplifier which is activated only in the presence of virus by aptamer recognition. The reaction process was carried out though one-pot method at 37 °C, which can save time and resources. In addition, magnetic beads used in reaction system can simplify operation, as well as provide ideas for developing biosensing robots via magnetic field. This SERS aptasensor can detect SARS-CoV-2 virus with a LOD of 260 TU/µL within 40 min in the linear range of 625–10,000 TU/µL. Therefore, this convenience, speediness, sensitivity, and selectivity of detection has great prospects in analyzing SARS-CoV-2 viral particles or other viruses in environment as well as monitoring of environmental virus sources.

Comparison of ADVIA Centaur ultra-sensitive and high-sensitive assays for troponin I in serum

Cardiac troponin I (cTnI) is a standard biomarker for the diagnosis of acute myocardial infarction (AMI). While older, ultra-sensitive cTnI (us-cTnI) assays use the 99th percentile as the reference threshold, newer high-sensitive cTnI (hs-cTnI) assays use the limit of detection or functional sensitivity instead. However, little has been done to systematically compare these two methods. The present study also served as a validation of hs-cTnI in our laboratory. Here, we compared the results obtained from the blood serum obtained from 8810 patients using the us-cTnI and the hs-cTnI assays run in tandem on the ADVIA Centaur XP analyser. We found that in 2279 samples the concentration of cTnI measured with the ultra-sensitive method was below the detection limit, while with the high-sensitive method, only 540 were below the detection limit. We also compared results from these assays with the ultimate diagnosis of a subset of individuals. The analysis of the results below cut-off with the ultra-sensitive method showed that this method would not detect 96 cases related to heart disorder. Overall, the main finding of our research is that hs-cTnI is the preferable option and is able to be deployed effectively in the laboratory setting.

Circulating Exosome Cargoes Contain Functionally Diverse Cancer Biomarkers: From Biogenesis and Function to Purification and Potential Translational Utility.

Simple SummaryThe indolent nature of some cancers makes early detection challenging, as such significant effort is placed on identifying circulating cancer biomarkers using minimally invasive, highly sensitive diagnostic assays. Biological fluids contain small extracellular vesicles including exosomes, which have many tissue origins. Cancer cells increase production and release of exosomes in the circulation to deliver biologically active compounds that can reprogram recipient cells, which potentially represent a valuable source of biomarkers. In this review, we evaluate the biogenesis of exosomes to understand how cancer cells alter their production and how their molecular cargoes interact with the microenvironment. Next, we provide a comprehensive inventory of techniques available for exosome purification, as this represents the most critical aspect for sensitive and specific evaluation of their biomarker content. Finally, we provide a current evaluation of their use as human cancer biomarkers.Although diagnostic and therapeutic treatments of cancer have tremendously improved over the past two decades, the indolent nature of its symptoms has made early detection challenging. Thus, inter-disciplinary (genomic, transcriptomic, proteomic, and lipidomic) research efforts have been focused on the non-invasive identification of unique “silver bullet” cancer biomarkers for the design of ultra-sensitive molecular diagnostic assays. Circulating tumor biomarkers, such as CTCs and ctDNAs, which are released by tumors in the circulation, have already demonstrated their clinical utility for the non-invasive detection of certain solid tumors. Considering that exosomes are actively produced by all cells, including tumor cells, and can be found in the circulation, they have been extensively assessed for their potential as a source of circulating cell-specific biomarkers. Exosomes are particularly appealing because they represent a stable and encapsulated reservoir of active biological compounds that may be useful for the non-invasive detection of cancer. T biogenesis of these extracellular vesicles is profoundly altered during carcinogenesis, but because they harbor unique or uniquely combined surface proteins, cancer biomarker studies have been focused on their purification from biofluids, for the analysis of their RNA, DNA, protein, and lipid cargoes. In this review, we evaluate the biogenesis of normal and cancer exosomes, provide extensive information on the state of the art, the current purification methods, and the technologies employed for genomic, transcriptomic, proteomic, and lipidomic evaluation of their cargoes. Our thorough examination of the literature highlights the current limitations and promising future of exosomes as a liquid biopsy for the identification of circulating tumor biomarkers.

Ag@Pyc Nanocapsules as Electrochemiluminescence Emitters for an Ultrasensitive Assay of the APE1 Activity.

Herein, Ag@pyrenecarboxaldehyde nanocapsules (Ag@Pyc nanocapsules) as emitters were prepared to construct an ultrasensitive electrochemiluminescence (ECL) biosensor for the detection of the human apurinic/apyrimidinic endonuclease1 (APE1) activity. Ag nanoparticles on the surface of Pyc nanocapsules as coreaction accelerators could significantly promote coreactant peroxydisulfate (S2O82-) to generate massive reactive intermediates of sulfate radical anion (SO4•-), which interacted with the Pyc nanocapsules to achieve a strong ECL response. In addition, with the aid of APE1-triggered 3D DNA machine, trace target could be converted into a large number of mimic targets (MTs), which were positively correlated with the activity of APE1. Consequently, the proposed ECL biosensor realized an ultrasensitive detection of APE1 activity with an exceptional linear working range from 5 × 10-10 to 5 × 10-4 U·μL-1 and a lower limit of detection of 1.36 × 10-11 U·μL-1. This strategy provided a new approach to construct an efficient ternary system for the detection of biomolecules and early diagnosis of diseases.