Sensitive colorimetric assay using insulin G-quadruplex aptamer arrays on DNA nanotubes coupled with magnetic nanoparticles

Described here is a methodology for fabrication of a sensitive colorimetric nanoassay for measurement of insulin using G-quadruplex aptamer arrays on DNA nanotubes (DNTs) coupled with magnetic nanoparticles. The spectroscopic findings (e.g. visible spectra, velocity assay and limit of detection determination) indicated a highly sensitive performance of this new nanoassay in comparison to those results obtained from the insulin assay with non-arrayed aptamers. The clinical performance statistics (i.e. paired sample t -test, Bland–Altman plot and scatter diagram) from the newly developed assay and the enzyme-linked immunosorbent assay suggested its reliable precision and its acceptable repeatability for measurement of insulin in human sera. This is, to our knowledge, the first study for the application of magnetic nanoparticle-coupled DNTs for carrying G-quadruplex aptamers for detection of biomolecules (such as insulin) in human serum.

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Self-Assembly of Functional Nucleic Acid-Based Colorimetric Competition Assay for the Detection of Immunoglobulin E

Sensors ◽

10.3390/s19102224 ◽

2019 ◽

Vol 19 (10) ◽

pp. 2224 ◽

Cited By ~ 1

Author(s):

Xuexia Lin ◽

Caiyun Yu ◽

Honggui Lin ◽

Cui Wang ◽

Jianlong Su ◽

...

Keyword(s):

Self Assembly ◽

Immunoglobulin E ◽

Limit Of Detection ◽

Solid Phase ◽

Colorimetric Assay ◽

Cost Effective ◽

Enzyme Linked Immunosorbent Assay ◽

Cost Effective Approach ◽

G Quadruplex ◽

Functional Nucleic Acids

In this work, we have developed a simple and rapid colorimetric assay for the detection of immunoglobulin E (IgE) using functional nucleic acids (FNAs) and a solid-phase competition enzyme-linked immunosorbent assay (ELISA). The FNAs including aptamer of recombinant IgE, G-quadruplex and its complementary fragments were immobilized on 96-well microplates to achieve recognition and detection of IgE in biological samples. The G-quadruplex DNAzyme catalyzed 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS)-hemin-H2O2 system was used to improve the sensitivity of colorimetric assay. In the presence of IgE, the hairpin structure and G-quadruplex would be destroyed, resulting in the inactivation of DNAzyme and subsequent reduction of its absorbance. This cost-effective approach detected IgE in the linear range from 5.0 pg/mL to 500 ng/mL, with the limit of detection (LOD) of 2.0 pg/mL, under optimal conditions. Moreover, the developed method was successfully applied to the rapid detection of IgE in human urine, indicating a great potentiality of this approach in clinical diagnosis and other biomedical applications.

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Ultrasensitive ELISA Developed for Diagnosis

10.20944/preprints201905.0328.v1 ◽

2019 ◽

Author(s):

Kanako Iha ◽

Mikio Inada ◽

Naoki Kawada ◽

Kazunari Nakaishi ◽

Satoshi Watabe ◽

...

Keyword(s):

Detection Method ◽

De Novo ◽

Limit Of Detection ◽

Colorimetric Assay ◽

Nucleic Acid Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Medical Facilities ◽

Ultrasensitive Assay ◽

Maximum Absorption Wavelength ◽

Hiv 1

For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This ultrasensitive assay consists of sandwich enzyme-linked immunosorbent assay (ELISA) and thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the accumulated thio-NADH is measured at its maximum absorption wavelength of 400 nm. We have successfully achieved a limit of detection of ca. 10–18 moles/assay for a target protein. As an example of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10−18 moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 10−19 moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary adiponectin. This method is highly versatile, because simply changing the antibody enables the detection of various proteins. This assay system requires only the measurement of absorbance, thus it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non-amplification nucleic acid detection method for nucleic acids using hybridization. These de novo methods will enable simple, rapid, and accurate diagnosis.

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Ultrasensitive ELISA Developed for Diagnosis

Diagnostics ◽

10.3390/diagnostics9030078 ◽

2019 ◽

Vol 9 (3) ◽

pp. 78 ◽

Cited By ~ 7

Author(s):

Kanako Iha ◽

Mikio Inada ◽

Naoki Kawada ◽

Kazunari Nakaishi ◽

Satoshi Watabe ◽

...

Keyword(s):

Detection Method ◽

De Novo ◽

Limit Of Detection ◽

Colorimetric Assay ◽

Nucleic Acid Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Medical Facilities ◽

Ultrasensitive Assay ◽

Maximum Absorption Wavelength ◽

Hiv 1

For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This ultrasensitive assay consists of a sandwich enzyme-linked immunosorbent assay (ELISA) and thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the accumulated thio-NADH is measured at its maximum absorption wavelength of 405 nm. We have successfully achieved a limit of detection of ca. 10−18 moles/assay for a target protein. As an example of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10−18 moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 10−19 moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary adiponectin. This method is highly versatile because simply changing the antibody enables the detection of various proteins. This assay system requires only the measurement of absorbance, thus it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non-amplification nucleic acid detection method for nucleic acids using hybridization. These de novo methods will enable simple, rapid, and accurate diagnosis.

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Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

10.26434/chemrxiv.5662315.v1 ◽

2017 ◽

Author(s):

Bo Tian ◽

Peter Svedlindh ◽

Mattias Strömberg ◽

Erik Wetterskog

Keyword(s):

Magnetic Nanoparticles ◽

Ferromagnetic Resonance ◽

Limit Of Detection ◽

Point Of Care ◽

Rolling Circle Amplification ◽

Resonance Field ◽

Isothermal Amplification ◽

Detection Sensitivity ◽

Serum Samples ◽

Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, i.e., rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg2P2O7 (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.

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Pitfalls in the Immunochemical Determination of β‑Lactam Antibiotics in Water

Antibiotics ◽

10.3390/antibiotics10030298 ◽

2021 ◽

Vol 10 (3) ◽

pp. 298

Author(s):

Alexander Ecke ◽

Rudolf J. Schneider

Keyword(s):

Limit Of Detection ◽

Cost Effective ◽

Enzyme Linked Immunosorbent Assay ◽

Degree Of Hydrolysis ◽

Site Analysis ◽

Antibody Recognition ◽

Immunochemical Determination ◽

Key Factor ◽

Lactam Ring

Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β‑lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.

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Phthalocyanine-Functionalized Magnetic Silica Nanoparticles as Anion Chemosensors

Sensors ◽

10.3390/s21051632 ◽

2021 ◽

Vol 21 (5) ◽

pp. 1632

Author(s):

João M. M. Rodrigues ◽

Andreia S. F. Farinha ◽

Zhi Lin ◽

José A. S. Cavaleiro ◽

Augusto C. Tome ◽

...

Keyword(s):

Magnetic Nanoparticles ◽

Dimethyl Sulfoxide ◽

Silica Nanoparticles ◽

Hybrid Material ◽

Limit Of Detection ◽

Active Material ◽

Anion Detection ◽

Anionic Species ◽

Magnetic Silica ◽

Magnetic Silica Nanoparticles

Anionic species are one of the most common pollutants in residual and freshwaters. The presence of anthropogenic anions in water drastically increases the toxicity to living beings. Here, we report the preparation of a new optical active material based on tri(tosylamino)phthalocyanines grafted to ferromagnetic silica nanoparticles for anion detection and removal. The new unsymmetrical phthalocyanines (Pcs) proved to be excellent chemosensors for several anions (AcO−, Br−, Cl−, CN−, F−, H2PO4−, HSO4−, NO2−, NO3−, and OH−) in dimethyl sulfoxide (DMSO). Furthermore, the Pcs were grafted onto magnetic nanoparticles. The resulting novel hybrid material showed selectivity and sensitivity towards CN−, F−, and OH− anions in DMSO with limit of detection (LoD) of ≈4.0 µM. In water, the new hybrid chemosensor demonstrated selectivity and sensitivity for CN− and OH− anions with LoD of ≈0.2 µM. The new hybrids are easily recovered using a magnet, allowing recyclability and reusability, after acidic treatment, without losing the sensing proprieties.

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Analytical assessment of ortho clinical diagnostics high-sensitivity cardiac troponin I assay

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-1115 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Peter A. Kavsak ◽

Tara Edge ◽

Chantele Roy ◽

Paul Malinowski ◽

Karen Bamford ◽

...

Keyword(s):

Cardiac Troponin ◽

Troponin I ◽

Limit Of Detection ◽

Clinical Performance ◽

Characteristic Curve ◽

Area Under The Curve ◽

High Sensitivity ◽

Cardiac Troponin I ◽

Clinical Diagnostics ◽

Ortho Clinical Diagnostics

AbstractObjectivesTo analytically evaluate Ortho Clinical Diagnostics VITROS high-sensitivity cardiac troponin I (hs-cTnI) assay in specific matrices with comparison to other hs-cTn assays.MethodsThe limit of detection (LoD), imprecision, interference and stability testing for both serum and lithium heparin (Li-Hep) plasma for the VITROS hs-cTnI assay was determined. We performed Passing-Bablok regression analyses between sample types for the VITROS hs-cTnI assay and compared them to the Abbott ARCHITECT, Beckman Access and the Siemens ADVIA Centaur hs-cTnI assays. We also performed Receiver-operating characteristic curve analyses with the area under the curve (AUC) determined in an emergency department (ED)-study population (n=131) for myocardial infarction (MI).ResultsThe VITROS hs-cTnI LoD was 0.73 ng/L (serum) and 1.4 ng/L (Li-Hep). Stability up to five freeze-thaws was observed for the Ortho hs-cTnI assay, with the analyte stability at room temperature in serum superior to Li-Hep with gross hemolysis also affecting Li-Hep plasma hs-cTnI results. Comparison of Li-Hep to serum concentrations (n=202), yielded proportionally lower concentrations in plasma with the VITROS hs-cTnI assay (slope=0.85; 95% confidence interval [CI]:0.83–0.88). In serum, the VITROS hs-cTnI concentrations were proportionally lower compared to other hs-cTnI assays, with similar slopes observed between assays in samples frozen <−70 °C for 17 years (ED-study) or in 2020. In the ED-study, the VITROS hs-cTnI assay had an AUC of 0.974 (95%CI:0.929–0.994) for MI, similar to the AUCs of other hs-cTn assays.ConclusionsLack of standardization of hs-cTnI assays across manufacturers is evident. The VITROS hs-cTnI assay yields lower concentrations compared to other hs-cTnI assays. Important differences exist between Li-Hep plasma and serum, with evidence of stability and excellent clinical performance comparable to other hs-cTn assays.

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A Smartphone Camera Colorimetric Assay of Acetylcholinesterase and Butyrylcholinesterase Activity

Sensors ◽

10.3390/s21051796 ◽

2021 ◽

Vol 21 (5) ◽

pp. 1796

Author(s):

Miroslav Pohanka ◽

Jitka Zakova

Keyword(s):

Limit Of Detection ◽

Colorimetric Assay ◽

Point Of Care ◽

Specific Treatment ◽

Point Of Care Testing ◽

Analytical Instruments ◽

Colorimetric Test ◽

3D Printed ◽

Butyrylcholinesterase Activity ◽

Additional Education

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can serve as biochemical markers of various pathologies like liver disfunction and poisonings by nerve agents. Ellman’s assay is the standard spectrophotometric method to measure cholinesterase activity in clinical laboratories. The authors present a new colorimetric test to assess AChE and BChE activity in biological samples using chromogenic reagents, treated 3D-printed measuring pads and a smartphone camera as a signal detector. Multiwell pads treated with reagent substrates 2,6-dichlorophenolindophenyl acetate, indoxylacetate, ethoxyresorufin and methoxyresorufin were prepared and tested for AChE and BChE. In the experiments, 3D-printed pads containing indoxylacetate as a chromogenic substrate were optimal for analytical purposes. The best results were achieved using the red (R) channel, where the limit of detection was 4.05 µkat/mL for BChE and 4.38 µkat/mL for AChE using a 40 µL sample and a 60 min assay. The major advantage of this method is its overall simplicity, as samples are applied directly without any specific treatment or added reagents. The assay was also validated to the standard Ellman’s assay using human plasma samples. In conclusion, this smartphone camera-based colorimetric assay appears to have practical applicability and to be a suitable method for point-of-care testing because it does not require specific manipulation, additional education of staff or use of sophisticated analytical instruments.

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Interfacing aptamers, nanoparticles and graphene in a hierarchical structure for highly selective detection of biomolecules in OECT devices

Scientific Reports ◽

10.1038/s41598-021-88546-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Carlotta Peruzzi ◽

Silvia Battistoni ◽

Daniela Montesarchio ◽

Matteo Cocuzza ◽

Simone Luigi Marasso ◽

...

Keyword(s):

Biomedical Applications ◽

Au Nanoparticles ◽

Limit Of Detection ◽

High Sensitivity ◽

Minimal Invasiveness ◽

Hierarchic Structure ◽

Final Response ◽

Detection Of Biomolecules ◽

Good Signal ◽

Detection Of Biomarkers

AbstractIn several biomedical applications, the detection of biomarkers demands high sensitivity, selectivity and easy-to-use devices. Organic electrochemical transistors (OECTs) represent a promising class of devices combining a minimal invasiveness and good signal transduction. However, OECTs lack of intrinsic selectivity that should be implemented by specific approaches to make them well suitable for biomedical applications. Here, we report on a biosensor in which selectivity and a high sensitivity are achieved by interfacing, in an OECT architecture, a novel gate electrode based on aptamers, Au nanoparticles and graphene hierarchically organized to optimize the final response. The fabricated biosensor performs state of the art limit of detection monitoring biomolecules, such as thrombin-with a limit of detection in the picomolar range (≤ 5 pM) and a very good selectivity even in presence of supraphysiological concentrations of Bovine Serum Albumin (BSA-1mM). These accomplishments are the final result of the gate hierarchic structure that reduces sterich indrance that could contrast the recognition events and minimizes false positive, because of the low affinity of graphene towards the physiological environment. Since our approach can be easily applied to a large variety of different biomarkers, we envisage a relevant potential for a large series of different biomedical applications.

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Heat Inactivation of Different Types of SARS-CoV-2 Samples: What Protocols for Biosafety, Molecular Detection and Serological Diagnostics?

Viruses ◽

10.3390/v12070735 ◽

2020 ◽

Vol 12 (7) ◽

pp. 735 ◽

Cited By ~ 18

Author(s):

Boris Pastorino ◽

Franck Touret ◽

Magali Gilles ◽

Xavier de Lamballerie ◽

Remi N. Charrel

Keyword(s):

Infected Cell ◽

Limit Of Detection ◽

Elisa Test ◽

Cell Culture Supernatant ◽

Heat Inactivation ◽

Standard Precautions ◽

Viral Loads ◽

Human Sera ◽

European Norm ◽

C 30

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.

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