Characterization of nanozyme kinetics for highly sensitive detection.

Nanozymes have been widely used as enzyme substitutes. Based on a comprehensive literature survey of 261 publications, we report the significant differences in the Michaelis-Menten constants (Km) between peroxidase-mimicking nanozymes and horseradish peroxidase (HRP). Further, these differences were not considered in more than 60% of the publications for analytical developments. As a result, nanozymes' catalytic activity is limited, resulting in a potentially higher limit of detection (LOD). We used a peroxidase-mimicking Au@Pt nanozyme, which has Km for TMB comparable with HRP and three orders of magnitude higher Km for H2O2. Using the Au@Pt nanozyme as a label for immunoassays, non-optimized nanozyme substrate concentrations led to 30 times higher LOD compared to optimized conditions. The results confirm the necessity of measuring nanozymes' kinetic parameters and the corresponding adjustment of substrate concentrations for highly sensitive detection.

Nonsteady-State Electric Circuit in Electrophoresis on Paper: Thermal Consideration of Electrophoretic Lateral-Flow Assays.

Nonsteady-state behaviors are not expected in electric circuits that lack significant capacitance, inductivity, and/or active feedback. Here, we report that electrophoresis on paper─used, e.g., to electrophoretically driven lateral-flow immunoassays (LFIA)─can create a nonsteady-state electric circuit. We studied electrophoresis on 50 × 4 mm nitrocellulose membrane strips utilized in LFIA. The voltage was applied to strip termini immersed in reservoirs with a running buffer. If the electric power of this circuit exceeded approximately 0.5 W, neither the electric current nor the temperature map reached their steady states on a multiminute time scale. The current grew slowly to its maximum and then slowly decreased. The temperature map evolved slowly, with one or more hot spots appearing and disappearing gradually in different positions on the strip. The slow evolution of a temperature map led to the occurrence of a terminal hot spot in which the strip burned. No chaotic behavior was observed, i.e., time dependences of both the current and temperature map were reproducible. We analyzed major processes involved in paper-based electrophoresis and explained the nonsteady-state behavior. Unlike ordinary electric circuits with metal conductors, paper-based electrophoresis involves two slow processes: (i) intense buffer evaporation from hot spots and (ii) buffer supply from the reservoirs by an interplay of the capillary penetration and the electroosmotic flow. These processes affect heat generation and/or dissipation on the strip and, accordingly, the resistivity profile. The slow evolution of the resistivity profile is responsible for the nonsteady-state behavior. The results of our computer modeling support this explanation. The hot spots may have a destructive effect on electrophoretically driven LFIA. To avoid denaturation of immunoreagents, experimentalists should empirically confirm that spatiotemporal temperature maps are compatible with the developed assay.

Sensitive Immunochromatographic Determination of Salmonella typhimurium in Food Products Using Au@Pt Nanozyme.

In this study, we developed a sensitive immunochromatographic analysis (ICA) of the Salmonella typhimurium bacterial pathogen contaminating food products and causing foodborne illness. The ICA of S. typhimurium was performed using Au@Pt nanozyme as a label ensuring both colorimetric detection and catalytic amplification of the analytical signal due to nanozyme peroxidase-mimic properties. The enhanced ICA enabled the detection of S. typhimurium cells with the visual limit of detection (LOD) of 2 × 102 CFU/mL, which outperformed the LOD in the ICA with traditional gold nanoparticles by two orders of magnitude. The assay duration was 15 min. The specificity of the developed assay was tested using cells from various Salmonella species as well as other foodborne pathogens; it was shown that the test system detected only S. typhimurium. The applicability of ICA for the determination of Salmonella in food was confirmed in several samples of milk with different fat content, as well as chicken meat. For these real samples, simple pretreatment procedures were proposed. Recoveries of Salmonella in foodstuffs were from 74.8 to 94.5%. Due to rapidity and sensitivity, the proposed test system is a promising tool for the point-of-care control of the Salmonella contamination of different food products on the whole farm-to-table chain.

Post-Assay Chemical Enhancement for Highly Sensitive Lateral Flow Immunoassays: A Critical Review.

Lateral flow immunoassay (LFIA) has found a broad application for testing in point-of-care (POC) settings. LFIA is performed using test strips-fully integrated multimembrane assemblies containing all reagents for assay performance. Migration of liquid sample along the test strip initiates the formation of labeled immunocomplexes, which are detected visually or instrumentally. The tradeoff of LFIA's rapidity and user-friendliness is its relatively low sensitivity (high limit of detection), which restricts its applicability for detecting low-abundant targets. An increase in LFIA's sensitivity has attracted many efforts and is often considered one of the primary directions in developing immunochemical POC assays. Post-assay enhancements based on chemical reactions facilitate high sensitivity. In this critical review, we explain the performance of post-assay chemical enhancements, discuss their advantages, limitations, compared limit of detection (LOD) improvements, and required time for the enhancement procedures. We raise concerns about the performance of enhanced LFIA and discuss the bottlenecks in the existing experiments. Finally, we suggest the experimental workflow for step-by-step development and validation of enhanced LFIA. This review summarizes the state-of-art of LFIA with chemical enhancement, offers ways to overcome existing limitations, and discusses future outlooks for highly sensitive testing in POC conditions.

Electrophoretic Assembly of Antibody-Antigen Complexes Facilitates 1000 Times Improvement in the Limit of Detection of Serological Paper-Based Assay.

Serological assays detect the presence of specific antibodies in blood. There are urgent and important applications for serological point-of-care (POC) assays. However, available detection methods are either insufficiently sensitive or too complex for POC settings. Here, we demonstrate that lateral flow immunoassay (LFIA), which is arguably the simplest universal molecular detection approach, can serve as a methodological platform for highly sensitive serological POC assays if combined with a simple, fast, and inexpensive electrophoretic step. In this work, we compared such electrophoretically driven LFIA (eLFIA) with conventional LFIA for the detection of immunoglobulins G against hepatitis B and C in serum. The limit of detection of eLFIA was proven to be 1000 times lower than that of conventional LFIA and sufficiently low to support clinical serological tests. eLFIA takes less than 10 min, requires only a minor accessory powered by a small 9 V battery, and can be performed by an untrained person in the POC environment using a 3 µL specimen of finger-prick capillary blood.

Electrophoresis-Assisted Multilayer Assembly of Nanoparticles for Sensitive Lateral Flow Immunoassay.

Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive point-of-need method. A major limitation of LFIA is a high limit of detection (LOD), which impacts its diagnostic sensitivity. To overcome this limitation, we introduce a signal-enhancement procedure that is performed after completing LFIA and involves controllably moving biotin- and streptavidin-functionalized gold nanoparticles by electrophoresis. The nanoparticles link to immunocomplexes forming multilayer aggregates on the test strip, thus, enhancing the signal. Here, we demonstrate lowering the LOD of hepatitis B surface antigen from approximately 8 to 0.12 ng mL-1 , making it clinically acceptable. Testing 118 clinical samples for hepatitis B showed that signal enhancement increased the diagnostic sensitivity of LFIA from 73 % to 98 % while not affecting its 95 % specificity. Electrophoresis-driven enhancement of LFIA is universal (antigen-independent), takes two minutes, and can be performed by an untrained person.

Application of Au@Pt Nanozyme as Enhancing Label for the Sensitive Lateral Flow Immunoassay of Okadaic Acid.

In this study, a lateral flow immunoassay (LFIA) was developed to detect okadaic acid (OA) belonging to the diarrheic shellfish poisoning group of aquatic toxins. Newly obtained anti-OA monoclonal antibodies and bimetallic core@shell Au@Pt nanoparticles were used in the indirect format of the LFIA. Peroxidase-mimicking nanozyme properties of Au@Pt enabled using them to enhance band coloration on the test strips and, consequently, for increasing the LFIA sensitivity. The instrumental limit of detection (LOD), the working range of detectable concentrations, and the visual cutoff of the assay were 0.5, 0.8-6.8, and 10 ng/mL, respectively. The assay duration was 20 min. The rapid and simple sample preparation procedure was applied for seawater, river water, and fish samples. The total duration of the sample pretreatment and LFIA was 25/40 min for water/fish samples, ensuring testing rapidity. The developed test system provides sensitive control of raw materials and food products and can be used to detect OA at all stages of the food industry «from sea to fork¼ chains.

Comparative Study of In Situ Techniques to Enlarge Gold Nanoparticles for Highly Sensitive Lateral Flow Immunoassay of SARS-CoV-2.

Three techniques were compared for lowering the limit of detection (LOD) of the lateral flow immunoassay (LFIA) of the receptor-binding domain of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) based on the post-assay in situ enlargement of Au nanoparticles (Au NPs) on a test strip. Silver enhancement (growth of a silver layer over Au NPs-Au@Ag NPs) and gold enhancement (growth of a gold layer over Au NPs) techniques and the novel technique of galvanic replacement of Ag by Au in Au@Ag NPs causing the formation of Au@Ag-Au NPs were performed. All the enhancements were performed on-site after completion of the conventional LFIA and maintained equipment-free assay. The assays demonstrated lowering of LODs in the following rows: 488 pg/mL (conventional LFIA with Au NPs), 61 pg/mL (silver enhancement), 8 pg/mL (galvanic replacement), and 1 pg/mL (gold enhancement). Using gold enhancement as the optimal technique, the maximal dilution of inactivated SARS-CoV-2-containing samples increased 500 times. The developed LFIA provided highly sensitive and rapid (8 min) point-of-need testing.

Peroxidase-mimicking nanozyme with surface-dispersed Pt atoms for the colorimetric lateral flow immunoassay of C-reactive protein.

Platinum-containing nanozymes with peroxidase-mimicking activity (PMA) have found a broad application in bioanalytical methods and are potentially able to compete with enzymes as the labels. However, traditionally used methods for the synthesis of nanozymes result in only a small fraction of surface-exposed Pt atoms, which participate in catalysis. To overcome this limitation, we propose a new approach for the synthesis of nanozymes with the efficient dispersion of Pt atoms on particles' surfaces. The synthesis of nanozymes includes three steps: the synthesis of gold nanoparticles (Au NPs), the overgrowth of a silver layer over Au NPs (Au@Ag NPs, 6 types of NPs with different thicknesses of Ag shell), and the galvanic replacement of silver with PtCl62- leading to the formation of trimetallic Au@Ag-Pt NPs with uniformly deposited catalytic sites and high Pt-utilization efficiency. Au@Ag-Pt NPs (23 types of NPs with different concentrations of Pt) with various sizes, morphology, optical properties, and PMA were synthesized and comparatively tested. Using energy-dispersive spectroscopy mapping, we confirm the formation of core@shell Au@Ag NPs and dispersion of surface-exposed Pt. The selected Au@Ag-Pt NPs were conjugated with monoclonal antibodies and used as the colorimetric and catalytic labels in lateral flow immunoassay of the inflammation biomarker: C-reactive protein (CRP). The colorimetric signal enhancement was achieved by the oxidation of 3,3'-diaminobenzidine by H2O2 catalyzed by Au@Ag-Pt NPs directly on the test strip. The use of Au@Ag-Pt NPs as the catalytic label produces a 65-fold lower limit of CRP detection in serum (15 pg mL-1) compared with Au NPs and ensures the lowest limit of detection for equipment-free lateral flow immunoassays. The assay shows a high correlation with data of enzyme-linked immunosorbent assay (R2 = 0.986) and high recovery (83.7-116.2%) in serum and plasma. The assay retains all the benefits of lateral flow immunoassay as a point-of-care method.

The steadfast Au@Pt soldier: Peroxide-tolerant nanozyme for signal enhancement in lateral flow immunoassay of peroxidase-containing samples.

We report the approach for the detection of Au@Pt core@shell nanoparticles (nanozymes) with peroxidase-mimicking activity (PMA) in samples with high endogenous peroxidase activity (EPA). Unlike the endogenous peroxidases in plant extracts that are inhibited by elevated H2O2 (>20 mM), the PMA of nanozymes was stable in concentrated H2O2 (up to 4 M). Such a different stability of enzymes and Au@Pt to the substrate allowed for eliminating EPA and detecting only nanozymes. The developed approach was used for reaching a lower limit of detection (LOD) and eliminating the background for the lateral flow immunoassay (LFIA) of the important plant pathogen potato virus X (PVX) in leaf and tuber extracts. Using the PMA of Au@Pt, the LOD was reduced to 4 and 8 pg/mL in tuber and leaf extracts, respectively. The LOD values are 250- and 500-times lower in comparison with LFIA with conventional gold nanoparticles. The developed approach of peroxidases inhibition is universal for bioanalytical methods, and its applicability was confirmed by the elimination of EPA in three matrixes (serum, potato leaf and tuber extracts).

Urchin peroxidase-mimicking Au@Pt nanoparticles as a label in lateral flow immunoassay: impact of nanoparticle composition on detection limit of Clavibacter michiganensis.

The influence of Au@Pt nanoparticles' composition, morphology, and peroxidase-mimicking activity on the limit of detection (LOD) of lateral flow immunoassay (LFIA) has been investigated. Fourteen types of nanoparticles were synthesized by varying the concentration of Pt4+ (20-2000 µM), using gold nanoparticles (GNP, diameter 20.0 ± 2.6 nm) as the seeds and ascorbic acid as a reducing agent. Au@Pt nanoparticles and GNPs were conjugated with antibodies specific to the target analyte, a widespread and dangerous phytopathogenic bacteria species (Clavibacter michiganensis). We found that the 100-fold growth of the Pt4+ concentration was accompanied by an increase of the Au@Pt nanoparticle diameter (24-55 nm) and surface area with the formation of urchin-shaped morphology. These changes led to a 70-fold increase in peroxidase-mimicking activity in the solution (specific activity 0.06-4.4 U mg-1) and a 30-fold decrease in LOD using the catalytic activity of Au@Pt. The Au@Pt nanoparticles synthesized at 1000-2000 µM of Pt4+ demonstrated statistically indistinguishable catalytic activity. The highest sensitivity of LFIA was reached for Au@Pt nanoparticles synthesized at Pt4+ concentration equal to 1000 µM. Au@Pt nanoparticles saved most of their peroxidase-mimicking activity, whereas endogenous plant peroxidases were completely inhibited by sodium azide. The LOD of LFIA with Au@Pt nanoparticles synthesized at 1200 µM of Pt4+ was 300 colony-forming units (CFU) per mL of buffer and 500 CFU per mL of potato tuber extract, which provides 330- and 200-fold improvement compared to the conventional LFIA with GNPs. The assay consists of three rapid 5-min stages, namely, extraction, lateral flow, and color enhancement (oxidation of diaminobenzidine by Au@Pt nanoparticles). LFIA with the urchin Au@Pt nanoparticles allows the detection of latent bacterial infections rapidly without equipment or special skills. Graphical abstract.

Enlargement of Gold Nanoparticles for Sensitive Immunochromatographic Diagnostics of Potato Brown Rot.

Lateral flow immunoassay (LFIA) is a convenient tool for rapid field-based control of various bacterial targets. However, for many applications, the detection limits obtained by LFIA are not sufficient. In this paper, we propose enlarging gold nanoparticles' (GNPs) size to develop a sensitive lateral flow immunoassay to detect Ralstonia solanacearum. This bacterium is a quarantine organism that causes potato brown rot. We fabricated lateral flow test strips using gold nanoparticles (17.4 ± 1.0 nm) as a label and their conjugates with antibodies specific to R. solanacearum. We proposed a signal enhancement in the test strips' test zone due to the tetrachloroauric (III) anion reduction on the GNP surface, and the increase in size of the gold nanoparticles on the test strips was approximately up to 100 nm, as confirmed by scanning electron microscopy. Overall, the gold enhancement approach decreased the detection limit of R. solanacearum by 33 times, to as low as 3 × 104 cells∙mL⁻1 in the potato tuber extract. The achieved detection limit allows the diagnosis of latent infection in potato tubers. The developed approach based on gold enhancement does not complicate analyses and requires only 3 min. The developed assay together with the sample preparation and gold enlargement requires 15 min. Thus, the developed approach is promising for the development of lateral flow test strips and their subsequent introduction into diagnostic practice.

Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses.

This study presents new type of the lateral flow immunoassay (LFIA) for multi-target analysis. A test, named alarm-LFIA, has an essentially new function that consists in notice (signaling the danger) about the presence at least one target from the controlled list without identification. The design of the alarm-LFIA assumes one test zone, which contains a mixture of antibodies, and multi-specific conjugate that binds the several targets. The alarm test is based on the novel conjugate with broaden specificity due to the immobilisation of a mix of antibodies, specific to several structurally different targets, on the surface of gold nanoparticles. For proof of concept, multi-specific conjugate to five important potato viruses (potato virus X, -M, -S, -Y and potato leaf roll virus) was fabricated using five antibodies with different specificity. The alarm-LFIA was developed for rapid detection of the total infection caused by up to five viruses. Detection limits of the viruses in potato leaf extracts are from 10 to 30 ng/mL. The alarm-LFIA was successfully used for viruses' detection in potato leaves; results were confirmed by enzyme-linked immunosorbent assay. The proposed approach of alarm-LFIA shows great potential for the various cases when different targets of interest can occur simultaneously or separately in samples.

How to Improve Sensitivity of Sandwich Lateral Flow Immunoassay for Corpuscular Antigens on the Example of Potato Virus Y?

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) was used as the polyvalent antigen, which affects economically important plants in the Solanaceae family. The obtained polyclonal antibodies that are specific to PVY were characterized using a sandwich enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). For LFIA, the antibodies were conjugated with GNPs with a diameter of 17.4 ± 1.0 nm. We conducted LFIAs using GNP conjugates in a dried state on the test strip and after pre-incubation with a sample. Pre-incubating the GNP conjugates and sample for 30 s was found to decrease the detection limit by 60-fold from 330 ng∙mL-1 to 5.4 ng∙mL-1 in comparison with conventional LFIA. The developed method was successfully tested for its ability to detect PVY in infected and uninfected potato leaves. The quantitative results of the proposed LFIA with pre-incubation were confirmed by ELISA, and resulted in a correlation coefficient of 0.891. The proposed approach is rapid, simple, and preserves the main advantages of LFIA as a non-laboratory diagnostic method.

Post-assay growth of gold nanoparticles as a tool for highly sensitive lateral flow immunoassay. Application to the detection of potato virus X.

This article demonstrates a new kind of a highly sensitive lateral flow immunoassay (LFIA). It is based on the enlargement of the size of gold nanoparticles (GNPs) directly on the test strip after a conventional LFIA. Particle size enlargement is accomplished through the catalytic reduction of HAuCl4 in the presence of H2O2 and through the accumulation of additional gold on the surface of the GNPs. To attain maximal enhancement of the coloration of the zone in the test strip and to achieve a minimal background, the concentration of precursors, the pH value, and the incubation time were optimized. GNPs on the test strip are enlarged from 20 to 350 nm after a 1-min treatment at room temperature. The economically important and widespread phytopathogen potato virus X (PVX) was used as the target analyte. The use of the GNP enlargement method results in a 240-fold reduction in the limit of the detection of PVX, which can be as low as 17 pg·mL-1. The total duration of the assay, including virus extraction from the potato leaves, lateral flow, and the enhancement process, is only 12 min. The diagnostic efficiency of the technique was confirmed by its application to the analysis of potato leave samples. No false positives or false negatives were found. The technique does not depend on specific features of the target analyte, and it is conceivably applicable to numerous GNP-based LFIAs for important analytes. Graphical abstract An enlargement solution (containing HAuCl4 and H2O2) was dripped on the strip after common lateral flow immunoassay. Gold nanoparticles on the strip (20 nm) catalyze gold reduction and the formation of larger particles (up to 350 nm), resulting in a 240-fold lower detection limit within 1 min.

Double-enhanced lateral flow immunoassay for potato virus X based on a combination of magnetic and gold nanoparticles.

This study presents the joint use of magnetic nanoparticles (MNPs) and gold nanoparticles (GNPs) for double enhancement in a lateral flow immunoassay (LFIA). The study realizes two types of enhancement: (1) increasing the concentration of analytes in the samples using conjugates of MNPs with specific antibodies and (2) increasing the visibility of the label through MNP aggregation caused by GNPs. The proposed strategy was implemented using a LFIA for potato virus X (PVX), a significant potato pathogen. MNPs conjugated with biotinylated antibodies specific to PVX and GNPs conjugated with streptavidin were synthesized and characterized. The LFIAs with and without the proposed enhancements were compared. The double-enhanced LFIA achieved the highest sensitivity, equal to 0.25 ng mL-1 and 32 times more sensitivity than the non-enhanced LFIA (detection limit: 8 ng mL-1). LFIAs using one of the types of amplification (magnetic concentration without GNPs-causing aggregation or MNP aggregation without the concentration stage) showed intermediate levels of sensitivity. The double-enhanced LFIA was successfully used for PVX detection in potato leaves. The results for PVX detection in the infected plants were similar for the double-enhanced LFIA developed and the conventional LFIA based on the GNP conjugates; however, the new system provided significant coloring enhancement. This study confirmed that a simple combination of MNPs and GNPs has great potential for high-sensitivity detection and could possibly be adopted for LFIAs of other compounds.

Setting up the cut-off level of a sensitive barcode lateral flow assay with magnetic nanoparticles.

A barcode lateral flow immunoassay (LFIA) based on magnetic nanoparticles with a controllable cut-off level was developed for the first time. The regulation of the cut-off levels was based on the changes in the position and concentration of three antibodies (two monoclonal and polyclonal) with different affinities (KD=4.22×10-9, 9.67×10-9, 1.05×10-8 M), respectively. To obtain specific conjugates, monoclonal antibodies were covalently immobilized on the magnetic nanoparticles' surface. Potato virus X causing a reduction in the potato yield was used as a model polyvalent antigen. To detect potato virus X in the leaf extracts, a barcode LFIA with cut-off levels of 3, 30, and 150ng/mL was used for the analysis. The application of magnetic concentration leads to a six-fold reduction in the first cut-off level (0.5ng/mL) in comparison with magnetic LFIA without the concentration stage.

Enhancement of lateral flow immunoassay by alkaline phosphatase: a simple and highly sensitive test for potato virus X.

Alkaline phosphatase (ALP) was used as an amplification tool in lateral flow immunoassay (LFIA). Potato virus Ð¥ (PVX) was selected as a target analyte because of its high economic importance. Two conjugates of gold nanoparticles were applied, one with mouse monoclonal antibody against PVX and one with ALP-labeled antibody against mouse IgG. They were immobilized to two fiberglass membranes on the test strip for use in LFIA. After exposure to the sample, a substrate for ALP (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) was dropped on the test strip. The insoluble dark-violet diformazan produced by ALP precipitated on the membrane and significantly increased the color intensity of the control and test zones. The limit of detection (0.3 ng mL-1) was 27 times lower than that of conventional LFIA for both buffer and potato leaf extracts. The ALP-enhanced LFIA does not require additional preparation procedures or washing steps and may be used by nontrained persons in resource-limited conditions. The new method of enhancement is highly promising and may lead to application for routine LFIA in different areas. Graphical abstract Two gold nanoparticles (GNP) conjugates were used - the first with monoclonal antibodies (mAb) (GNP-mAb); the second - alkaline phosphatase-labeled antibody against mAb (GNP-anti-mAb-ALP). The immuno complexes are captured by the polyclonal antibodies (pAb) in the test zone. Addition of the substrate solution (BCIP/NBT) results in the accumulation of the insoluble colored product and in a significance increase in color intensity.

Unexpected Electrophoretic Behavior of Complexes between Rod-like Virions and Bivalent Antibodies.

Here we report on the unexpected electrophoretic behavior of complexes between rod-like virus particles (virions) and bivalent antibodies. The multiple complexes formed by the virions and antibodies migrated with electrophoretic mobilities of much greater absolute values than those of the unbound virions or antibodies while typically complexes have mobilities intermediate to those of their components. We hypothesized that the mobilities of unusually high absolute values are caused by the cross-linking of virions by bivalent antibodies into aggregates with prominent side-to-side binding. Theoretically, the mobility of such aggregates should be proportional to the square root of the number of cross-linked virions. The formation of virion aggregates with prominent side-to-side binding was confirmed by atomic force microscopy. The dependence of the aggregate mobility on the number of cross-linked virions can be used to estimate this number.

Complex analysis of concentrated antibody-gold nanoparticle conjugates' mixtures using asymmetric flow field-flow fractionation.

Conjugates of gold nanoparticles (GNPs) with antibodies are powerful analytical tools. It is crucial to know the conjugates' state in both the concentrated and mixed solutions used in analytical systems. Herein, we have applied asymmetrical flow field-flow fractionation (AF4) to identify the conjugates' state. The influence of a conjugate's composition and concentration on aggregation was studied in a true analytical solution (a concentrated mixture with stabilizing components). GNPs with an average diameter of 15.3±1.2nm were conjugated by adsorption with eight antibodies of different specificities. We found that, while the GNPs have a zeta potential of -31.6mV, the conjugates have zeta potentials ranging from -5.8 to -11.2mV. Increased concentrations (up to 184nM, OD520=80) of the mixed conjugate (mixture of eight conjugates) did not change the form of fractograms, and the peak areas' dependence on concentration was strongly linear (R2 values of 0.99919 and 0.99845 for absorption signal and light scattering, respectively). Based on the gyration (Rg) and hydrodynamic (Rh) radii measured during fractionation, we found that the nanoparticles were divided into two populations: (1) those with constant radii (Rg=9.9±0.9nm; Rh=14.3±0.5nm); and (2) those with increased radii from 9.9 to 24.4nm for Rg and from 14.3 to 28.1nm for Rh. These results confirm that the aggregate state of the concentrated and mixed conjugates' preparations is the same as that of diluted preparations and that AF4 efficiently characterizes the conjugates' state in a true analytical solution.