Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17β-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17β-estradiol is 1.9 pg mL⁻¹, which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.     

Dual-polarization interferometry for quantification of small molecules using aptamers

An interferometry-based method was developed for detection of a small molecule, argininamide. The quantification of argininamide was demonstrated using aptamers immobilized on silicone oxynitride sensor surface via avidin–biotin binding. The aptamers formed a thin film over avidin layer corresponding to a thickness of 1.2 nm, consistent with a molecular positioning of multipoint attachment to the surface. The binding of argininamide did not cause any significant changes in the thickness of the aptamer film, suggesting that the specific binding did not affect the overall conformation of the aptamer molecules after adaptive rearrangement of aptamer molecules. However, the binding results in clearly detectable changes in mass calculated from multiple parameters determined by mass deposition and structural changes. The limit of detection of the developed sensor was determined to be 5 μM. The sensor can monitor real-time changes in argininamide concentrations with high reliability and sensitivity. The model system demonstrated that a combined measurement considering structural and mass changes through interferometry-based techniques can overcome one of the major problems associated with real-time monitoring of small mass analytes.     

Direct detection of Δ9-tetrahydrocannabinol in aqueous samples using a homogeneous increasing fluorescence immunoassay (HiFi)

The detection of the major active component of cannabis, Δ9-tetrahydrocannabinol (THC), becomes increasingly relevant due to its widespread abuse. For control purposes, some easy-to-use, sensitive and inexpensive test methods are needed. We have developed a fluorescence immunoassay utilising THC–fluorescein conjugate as tracer. Fluorescence spectroscopy of the conjugate revealed an unusual property: The relatively weak fluorescence of a dilute tracer solution was increased by a factor of up to 5 after binding of a THC-specific antibody. Fluorescence lifetime measurements in aqueous solutions suggested two different tracer conformations both associated with quenching of fluorescein fluorescence by the intramolecular THC moiety. After antibody binding, the tracer enters a third conformation in which fluorescence quenching of fluorescein is completely suppressed. Utilising this property, we established a homogeneous competitive immunoassay (homogeneous increasing fluorescence immunoassay) with low detection limits. The test requires only two reagents, the new tracer molecule and an anti-THC antibody. A single test takes only 8 min. The dynamic detection range for THC is 0.5 to 20 ng/mL in buffer, with a limit of detection (LOD) of 0.5 ng/mL. The test also works in diluted saliva samples (1:10 dilution with buffer) with an LOD of 2 ng/mL and a dynamic range of 2–50 ng/mL.     

Lectin-modified piezoelectric biosensors for bacteria recognition and quantification

The use of lectins for microorganism biosensors fabrication is proposed. Lectins are immobilised onto a gold-plated quartz crystal for direct piezoelectric label-free transduction of the bacteria–lectin binding event using an electrochemical quartz crystal microbalance (EQCM). Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin immobilisation method and the biosensor performance. Adsorption on nonpolarised and polarised (−0.200 V) gold-coated quartz crystals and immobilisation through avidin–biotin binding were checked for Con A surface attachment. Lectin–bacteria binding was evaluated in all cases. With a crystal modified with Con A via avidin–biotin immobilisation we obtained a linear calibration plot between 5.0 × 10⁶ and 2.0 × 10⁷ cfu mL⁻¹ by measuring frequency changes with E. coli concentration 1 h after bacteria addition. A remarkable increase in sensitivity was achieved when the analytical solution contained free biotinylated Con A, as a consequence of multiple lectin adhesion to Escherichia coli cell wall, which produced an accumulation of Con A–E. coli conjugates in the form of multilayers at the electrode surface. A detection limit of approximately 1.0 × 10⁴ cfu mL⁻¹ was achieved. Moreover nonspecific adsorptions were minimised. Using Con A and lectin from Arachis hypogaea, different response profiles were achieved for Escherichia coli, Staphylococcus aureus and Mycobacterium phlei, thus demonstrating the feasibility of bacteria discrimination. An approach involving filtering of free and lectin-bound bacteria and introduction of a filter in the measuring cell allowed a significant frequency change to be obtained for an E. coli concentration of 1.0 × 10³ cfu mL⁻¹ in order to further increase the sensitivity and discriminate between viable and nonviable cells; an approach using electrochemical measurements of bacterial catalase activity was also checked.     

Biofunctional organic nanocrystals for quantitative detection of pathogen deoxyribonucleic acid

Advances in nanotechnology have had significant impacts in the field of biodiagnostics. In this study, we describe the novel application of dissolvable, organic and biofunctional nanocrystals for the quantitative detection of a PCR product. Fluorescein diacetate (FDA), a fluorogenic precursor of fluorescein, was milled in a solution of a polymeric surfactant to create a stable, nanosized colloid with an interface for coupling streptavidin molecules. The application of these particulate labels for the quantitative detection of biotinylated human papillomavirus (HPV) DNA, amplified in a standard PCR procedure, was demonstrated. After the affinity reaction, the FDA molecules were dissolved and concomitantly converted into fluorescein. This approach resulted in a high selectivity, short incubation times and a sensitivity up to 147 times greater than obtained from state-of-the-art, directly fluorescent-labeled streptavidins. This innovative method offers rapid detection of small amounts of nucleic acids because less target material and thus fewer PCR cycles are required.     

Characterization and application of quantum dot nanocrystal–monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot–MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen–antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC₅₀) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10–100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1–9.2%. Figure Shematic diagram of the direct cFLISA procedure Jianzhong Shen and Fei Xu contributed equally to this work.     

Rapid one-step whole blood C–reactive protein magnetic permeability immunoassay with monoclonal antibody conjugated nanoparticles as superparamagnetic labels and enhanced sedimentation

A rapid (5.5 min) one-step whole blood C–reactive protein (CRP) magnetic permeability immunoassay utilizing monoclonal antibody conjugated dextran iron oxide nanoparticles (70 nm) as superparamagnetic labels and mixed fractions (1:1 ratio of 15–40 and 60 μm) of polyclonal anti-CRP conjugated silica microparticles for enhanced sedimentation is described. In this one-step assay procedure, a whole blood sample (4 μl) is applied to an assay glass vial, containing both antibody conjugates, and mixed for 30 s. The target analyte, CRP, forms a sandwich complex between the conjugated nanoparticles and microparticles, and, subsequently, the complex sediments under normal gravitation within 5 min to the bottom of the vial. The magnetic permeability increase of the sediment due to the presence of the complexed superparamagnetic nanoparticles is determined using an inductance-based transducer. Assayed patient whole blood samples were compared with the Abbott Diagnostics Architect reference method. A strong linear correlation was observed for the CRP concentration range 0–260 mg/l in whole blood (y=1.001x+0.42, R ²=0.982, n=50). The CRP assay presented showed a limit of detection of 3 mg/l and a total imprecision (coefficient of variation) of 10.5%. On the basis of our observations, we propose a rapid, one-step, CRP assay for near-patient testing.     

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA–BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL⁻¹, and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74–110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL⁻¹ for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R ² = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples.     

Filtration-based tyramide amplification technique—a new simple approach for rapid detection of aflatoxin B<Subscript>1</Subscript>

The catalyzed reporter deposition (CARD) method of signal amplification, also called “tyramide signal amplification”, has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B₁ (AFB₁) in a variety of foodstuffs with a detection limit of 12.5 μg kg⁻¹. The assay procedure involves sequential addition of standards or sample, AFB₁–horseradish peroxidase (HRP) conjugate, B-T, avidin–HRP, and substrate solution over anti-AFB₁ antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB₁ at concentrations from 25 to 100 mg kg⁻¹ were between 95 and 105%. AFB₁ results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC. Figure The principle of filtration-based tyramide filtration technique     

Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout

Direct measurement of time-resolved fluorescence from a washed surface of an immunoassay well constitutes an advantage compared with label development options involving signal generation in solution. Epi-fluorometric detection collects the signal from only a small part of the microtiter well’s bottom surface and it is inadequate for the optimal assay sensitivity when using binding surfaces introduced by large coating volume. This study reports on the use of streptavidin-coated spots intended to condense the binding of the labeled antibodies to coincide with the excitation beam. The spots were generated in special microtiter wells containing 2.5-mm, 3.5-mm, and 4.5-mm diameter indentations by adsorption from liquid droplets containing either native (SAv) or modified high-capacity (GA-SAv) streptavidin. The SAv-coated and GA-SAv-coated spots exhibited maximum Eu–biotin binding densities of 0.080 and 0.47 pmol/mm², respectively. A sandwich-type immunoassay of thyroid-stimulating hormone (TSH) provided a fivefold to sixfold increase in the signal-to-background ratios of the spot assay and an equivalent improvement in the detection limit (DL < 0.01 mU/L) compared with a reference assay. Figure The condensation of the binding area into a spot (right) results in a denser collection of the labeled antibodies and more favorable signal-to-background ratios compared with a regular approach using a large binding area (left)     

Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay

Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L⁻¹ and 1.3 ng L⁻¹ using two different up-converting phosphors and 0.16 ng L⁻¹ using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.     

Preparation of polypseudorotaxane consisting of fluorescent molecule-modified β-cyclodextrins and biotin-terminated poly(propylene glycol) with high yield

A polypseudorotaxane was prepared using 4-(N,N-dimethylamino) benzoyl modified β-cyclodextrin (DMAB-β-CD) and biotin-terminated poly(propylene glycol) (biotin-terminated PPG) in water for 7 days. When the biotin-terminated PPG was added to a saturated solution of DMAB-β-CD at room temperature, the polypseudorotaxane was not obtained. However, with increasing temperature to 60 °C, precipitation was observed. The ¹H-NMR spectrum indicated that the obtained precipitate was the polypseudorotaxane. The final yield was 54%, which was higher than that previously reported for polypseudorotaxane formation using chemically modified β-CD. The polypseudorotaxane can be useful to make a polyrotaxane via avidin–biotin molecular recognition using the terminal biotin group.     

Flow injection chemiluminescence immunoassay for 17β-estradiol using an immunoaffinity column

A new flow injection chemiluminescent immunoassay was developed for the detection of 17β-estradiol (E₂). The method uses p–iodophenol (PIP) as enhancer and is based on a solid-phase immunoassay format in which an E₂–OVA immobilized immunoaffinity column inserted in the flow system is used to trap unbound horseradish peroxidase (HRP)-labeled anti-E₂ antibody after an off-line incubation of E₂ with HRP-labeled anti-E₂ antibody. The trapped enzyme conjugate was detected by injecting substrates to produce an enhanced chemiluminescence (CL) response. The linear range for E₂ was 10.0–1,000.0 ng mL⁻¹ with a correlation coefficient of 0.996 and a detection limit of 3.0 ng mL⁻¹. The sampling and chemiluminescence detection time for one sample was 400 s after a pre-incubation procedure of 30 min. Serum samples detected by this method were in good agreement with the results obtained by EIA with E₂–biotin.      

Sensitive miniature single-particle immunoassay of prostate-specific antigen using time-resolved fluorescence

The present study describes the development of a quantitative miniaturized single microparticle immunoassay. The main objective of the study was to evaluate the performance of a miniature heterogeneous immunoassay on a single microparticle in respect to assay kinetics, volume, and sensitivity, binding capacity of microparticles and sensitivity using europium(III) nanoparticle labels. The performance of the single microparticle assay of prostate-specific antigen (PSA) was investigated using different-sized microparticles (60-920 μm in diameter) and microtiter well as a solid-phase. Equilibration time of the assay was shown to be dependent in a linear manner on surface-to-volume ratio, i.e. larger surface-to-volume translated to a faster reaction. However, no correlation between PSA binding capacity and equilibration time was observed in these kinetic studies. Only moderate improvement in assay kinetics was found when PSA binding capacity was increased on a microparticle. Using europium(III) nanoparticle labels, 107 nm in diameter, coated with streptavidin a detection sensitivity of 30 ng l⁻¹ (0.1 amol) was achieved in 1 μl total assay volume per microparticle. This was 50-fold higher compared to the same assay performed with intrinsically fluorescent europium(III) labels.     

Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen

A micro-magnetic chemiluminescence (CL) enzyme immunoassay with high sensitivity, selectivity, and reproducibility was developed for the determination of the tumor marker, carcinoembryonic antigen (CEA) in human serum. A sandwich scheme assay has been utilized with fluorescein isothiocyanate antibody (FITC)-labeled anti-CEA antibody and alkaline phosphate (ALP)-labeled anti-CEA antibody being used in the CL detection. The CL signal produced by the emission of photons from 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) was directly proportional to the amount of analyte present in a sample solution. The influences of the reaction time of antigen with antibody, the reaction time of substrate with label, the dilution ratio of ALP-labeled anti-CEA antibody, the concentration of FITC-labeled anti-CEA antibody, and other relevant variables upon the CL signal were examined and optimized. The CL responses depended linearly on the CEA concentration over the range from 2 to 162 ng mL⁻¹ in a logarithmic plot. Assay sensitivity as low as 0.69 ng mL⁻¹ was achieved. A coefficient of variance of less than 13% was obtained for intra- and inter-assay precision. This method has been successfully applied to the analysis of CEA in human serum. According to the procedure based on spiked standards, the recoveries obtained were 80–110%. Comparison experiments were carried out with the commercially available CEA chemiluminescence immunoassay. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level).     

Fluorescent organic nanocrystal confined in sol–gel matrix for bio-imaging

The sol–gel chemistry combined to a spray-drying process allowed us to control the formation of original hybrid core–shell nanoparticles constituted by molecular nanocrystals of rubrene embedded in biocompatible silicate spheres. With a good management of all the physical (gas flows, temperatures) and chemical (dye, solvent and alkoxide natures, concentrations, and hydrolysis and condensation conditions) parameters, we optimized a one-step and self-assembly process allowing to obtain nanoparticles exhibiting promising optical properties such as highly fluorescent labels (two-photon excitation) for medical imaging. Moreover, the presence of Si–OH functions on the silicate shell surface make easy to functionalize these fluorescent nanoparticles by grafting biomolecules for targeting properties. The confined nucleation and growth of rubrene nanocrystals in sol–gel silicate spheres during their drying in the air laminar flows, prevents any phase segregation or particle coalescence and stabilizes mechanically and chemically the organic cores. The first particle sizes obtained in these first experiments are ranging between 80 and 600 nm, but lower diameters will be easily prepared by increasing the solvent amount. Transmission electron microscopy was used to characterize the rubrene organic cores. The electron diffraction patterns performed at 100 K, under low-dose illumination to avoid amorphization of the samples during electron irradiation, have shown the good crystallinity of the NP rubrene cores that seem to be constituted by single rubrene nanocrystals. Finally, optical confocal microscopy, used in reflection and fluorescence modes, showed that all the core–shell particles are strongly fluorescent. This high fluorescence intensity arises from the high molecule numbers of rubrene nanocrystals, which enhance the absorption and emission cross sections.     

Fluorescence signal transduction mechanism for immunoassay based on zinc ion release from ZnS nanocrystals

In this work, a fluorescence signal transduction mechanism based on cation release from ZnS nanocrystals was developed for sandwich immunoassay. In this mechanism, ZnS nanocrystals as labels in immunoassay are dissolved by acid to release zinc ions. After pH adjustment of the dissolving solution using a basic solution, zinc-ion sensitive fluorescence indicator Fluozin-3 is added to bind with the released zinc ions for sensitive fluorescence measurement. Using mouse IgG as a model analyte, the immunoassay adopting this signal transduction mechanism demonstrates a low detection limit around 1 pM and a detection range with two orders of magnitude (1 pM to 0.5 nM).     

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles’ colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core–shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient’s sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.     

A highly sensitive detection platform based on surface-enhanced Raman scattering for Escherichia coli enumeration

A very sensitive and highly specific heterogeneous immunoassay system, based on surface-enhanced Raman scattering (SERS) and gold nanoparticles, was developed for the detection of bacteria and other pathogens. Two different types of gold nanoparticles (citrate-stabilized gold nanosphere and hexadecyltrimethylammonium bromide (CTAB)-stabilized gold nanorod particles) were examined and this immunoassay was applied for the detection of Escherichia coli. Raman labels were constructed by using these spherical and rod-shaped gold nanoparticles which were first coated with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and subsequently with a molecular recognizer. The working curve was obtained by plotting the intensity of the SERS signal of the symmetric NO₂ stretching of DTNB at 1,333 cm⁻¹ versus the concentration of the E. coli. The analytical performance of gold particles was evaluated via a sandwich immunoassay, and linear calibration graphs were obtained in the E. coli concentration range of 10¹–10⁵ cfu/mL with a 60-s accumulation time. The sensitivity of the Raman label fabricated with gold nanorods was more than three times higher than spherical gold nanoparticles. The selectivity of the developed sensor was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the developed immunoassay to detect E. coli in real water samples was also demonstrated.     

Colorimetric aggregation assay based on array of gold and silver nanoparticles for simultaneous analysis of aflatoxins,ochratoxin and zearalenone by using chemometric analysis and paper based analytical devices

A fluorometric assay was introduced to determine Acinetobacter baumannii (A. baumannii) in blood samples by utilizing Zr-MOFs both as functional coating for magnetic Fe3O4 nanoparticles to provide modification surface (Zr-mMOF) and as fluorescein carrier to produce fluorescence signals (F@UIO-66-NH2). Through strong Zr-O-P bonding, two distinct terminal phosphate-labeled A. baumannii and lipopolysaccharide (LPS) specific aptamers were attached onto Zr-MOFs to fabricate the magnetic core-shell capture probe (denoted as Zr-mMOF-p-Ab-Apt) and signal probe (denoted as F@UIO-66-NH2-p-LPS-Apt), respectively. After successive incubation with A. baumannii in blood samples and magnetic separation, the sandwich-type composite of capture probe/A. baumannii cells/signal probe was treated with high concentration of anionic phosphate ions to destroy the nano-structure of UIO-66-NH2 in the signal probe and fast release of fluorescein to produce amplified fluorescence signals. Due to the high aptamer modification efficiency of Zr-mMOF-p-Ab-Apt (up to 93%) and its strong affinity to A. baumannii, the enrichment efficiency of this capture probe has reached to 96.7%. Further, due to the high fluorescein loading efficiency of UIO-66-NH2 and our novel amplification strategy to destroy F@UIO-66-NH2-p-LPS-Apt to release and amplify fluorescein signals at 512 nm in the presence of high concentration of anionic phosphate ions, the sensitivity of this method has reached 10 cfu mL−1. This method allows enrichment and determination of A. baumannii within ~2.5 h. The limit of detection of A. baumannii in blood samples is 10 cfu mL−1 with a linear range of 101–105 cfu mL−1. This indicates the potential of this assay for diagnosis of bloodstream infection in early stage.