Isocratic reporter-exclusion immunoassay using restricted-access adsorbents.

We introduce analyte-dependent exclusion of reporter reagents from restricted-access adsorbents as the basis of an isocratic reporter-exclusion immunoassay for viruses, proteins, and other analytes. Capto™ Core 700 and related resins possess a noninteracting size-selective outer layer surrounding a high-capacity nonspecific mixed-mode capture adsorbent core. In the absence of analyte, antibody-enzyme reporter conjugates can enter the adsorbent and be captured, and their signal is lost. In the presence of large or artificially-expanded analytes, reporter reagents bind to analyte species to form complexes large enough to be excluded from the adsorbent core, allowing their signal to be observed. This assay principle is demonstrated using M13 bacteriophage virus and human chorionic gonadotropin as model analytes. The simple isocratic detection approach described here allows a rapid implementation of immunoassay for detection of a wide range of analytes and uses inexpensive, generally-applicable, and stable column materials instead of costly analyte-specific immunoaffinity adsorbents.

Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays.

We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ∼100 times lower than those of previously reported IgE assays.

The effect of polarity and hydrogen bonding on the electronic and vibrational structure of the salicylate anion in acetonitrile and water: implicit and explicit solvation approaches.

This study investigates the fluorescence quenching of the salicylate anion in water compared to acetonitrile (ACN) and the stability of its keto structure in ACN using DFT and TD-DFT methods at the 6-311++G(d,p) basis set. Computational simulations in implicit and explicit environments of ACN and water reveal the effects of solvent polarity and hydrogen bonding on enol [Od-H⋯Oa] and keto [Od⋯H-Oa] tautomerization, fluorescence quenching, and the spectral profile of the salicylate anion. Implicit solvation models show a barrier height of approximately 1.9 kcal mol-1 in ACN and 3.6 kcal mol-1 in water for enol-keto tautomerization in the ground state, with no barrier in the excited state, leading to an ESIPT reaction in both solvents, but only ground state proton transfer in ACN. Simulated absorption spectra for both enol and keto forms are similar in both solvents, while the emission spectrum is red-shifted in water. Explicit solvation studies indicate greater stabilization of the salicylate anion in water than in ACN, with a blue shift in absorption and emission spectra and varying oscillator strengths. Solvent molecule positioning affects enol-keto stabilization in the ground state, but only the keto structure is stabilized in the excited state. Simulated IR spectra in water show a blue shift in Od-H stretching frequency and increased water molecule vibrational frequencies, suggesting non-radiative excitation energy transfer from salicylate ions to water molecules via n → σ* intermolecular hydrogen bonding interactions. This mechanism explains the fluorescence quenching observed in water and results align with experimental data, indicating hydrogen-bonded keto form stabilization both in water and ACN.

Functionalized viral nanoparticles as ultrasensitive reporters in lateral-flow assays.

Two types of viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). The particles were successfully integrated into an immunochromatographic assay detecting MS2 bacteriophage, a model for viral pathogens. The sensitivity of the assay was greatly superior to conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments.

Spectral and time domain fluorescence spectroscopy of gentisic acid molecule in protic and aprotic polymer matrix.

In the present work, the effect of polymer microenvironment on the photophysics of gentisic acid molecule [2,5-dihydroxybenzoic acid] (GA), steady-state and time-domain fluorescence measurements at different pH conditions were carried out in protic [polyvinyl alcohol PVA] and aprotic [polymethyl methacrylate (PMMA)] polymer matrices. Change in the proticity of the microenvironment of the polymer traps different ionic species along with the neutral form of rotamer P and R conformers of GA molecule, are found to be responsible for the change in the spectral, multi-exponential decay behaviour. In protic polymer, the appearance of a single emission band indicates, dissociation of the GA molecule is very high, and it present as a monoanion along with hydrogen-bonded P and R rotamers. However, in the basic polymer film, most of the conformers of R converted to the anion. In contrast, protonation slows down the dissociation of both P and R forms in the acidic film. Unlike PVA matrix, in PMMA, dual emission band appears due to slow dissociation of GA molecule and hydrogen-bonded rotamer P, and R form exists with monoanion species. The magnitude of large stokes shifted red emission due to excited-state intramolecular proton transfers (ESIPT) found grater in rotamer P compared to its anionic species (green emission) and a blue emission corresponds to rotamer R.

Sensitive detection of norovirus using phage nanoparticle reporters in lateral-flow assay.

Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair.

Detection of viruses by counting single fluorescent genetically biotinylated reporter immunophage using a lateral flow assay.

We demonstrated a lateral flow immunoassay (LFA) for detection of viruses using fluorescently labeled M13 bacteriophage as reporters and single-reporter counting as the readout. AviTag-biotinylated M13 phage were functionalized with antibodies using avidin-biotin conjugation and fluorescently labeled with AlexaFluor 555. Individual phage bound to target viruses (here MS2 as a model) captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses.

Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus.

Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for "point of care" screening of BKV.

Effect of concentration and wavelength of excitation on the photophysics of salicylate anion in acetonitrile and water.

Sodium salicylate (NaSA) molecule exists as salicylate anion in acetonitrile (ACN) and water solvents, and exhibits large Stokes shifted fluorescence due to excited state intramolecular proton transfer (ESIPT), with decay times of âˆ¼5 ns in ACN and âˆ¼3.9 ns in water by 300 nm (absorption maxima) excitation. Previous studies report both ground and excited state enol-keto tautomerization in ACN, but only excited state tautomerization in water at 10-4 M. However, the current work explores the effect of concentration and excitation wavelengths on the photoinduced dynamics of ESIPT in the salicylate anion. On increasing the concentration of NaSA, no change in absorption spectra appears in both the solvents, and emission spectra of NaSA in water remain unaffected by changes in concentration or excitation wavelength, whereas, a slight red shift and decrease in FWHM appears in ACN. Time-domain fluorescence measurements show predominantly single-exponential decay throughout the emission profile by 300 nm excitation above the 10-5 M concentration in both the solvents, while by 375 nm excitation, multi-exponential fluorescence decay is observed at low concentrations, and as the concentration of NaSA increases, this decay behaviour tends to converge towards a single exponential decay. These results suggest that solute-solvent interactions stabilize the ground-state intermolecular hydrogen-bonded species at low concentrations, while higher concentrations weaken these interactions, leading to emission solely from the salicylate anion. Peak fit analysis of excitation spectra confirms enol-keto tautomerization in both the solvents, with the keto form being more stabilized in ACN. These findings underscore that in ACN, behaviour of NaSA is influenced by both concentration and excitation wavelength and contrary to previous reports, the keto form of the molecule is also present in water, though at a very low concentration and an increase in non-radiative transitions in water cause fluorescence quenching.