Merging Lateral Flow Immunoassay with Electroanalysis as a Novel Sensing Platform: Prostate Specific Antigen Detection as Case of Study.

The COVID-19 pandemic highlighted lateral flow immunoassay (LFIA) strips as the most known point-of-care (POC) devices enabling rapid and easy detection of relevant biomarkers by nonspecialists. However, these diagnostic tests are usually associated with the qualitative detection of the biomarker of interest. Alternatively, electrochemical-based diagnostics, especially known for diabetes care, enable quantitative determination of biomarkers. From an analytical point perspective, the combination of the two approaches might represent a step forward for the POC world: in fact, electrochemical transduction is attractive to be integrated into LFIA strips due to its simplicity, high sensitivity, fast signal generation, and cost effectiveness. In this work, a LFIA strip has been combined with an electrochemical transduction, yielding an electrochemical LFIA (eLFIA). As a proof-of-concept method, the detection of prostate-specific antigen has been carried out by combining a printed-electrochemical strip with the traditional LFIA tests. The electrochemical detection has been based on the measurement of Au ions produced from the dissolution of the gold nanoparticles previously captured on the test line. The analytical performances obtained at LFIA and eLFIA were compared, highlighting how the use of differential pulse voltammetry allowed for a lower detection limit (2.5-fold), respectively, 0.38 and 0.15 ng/mL, but increasing the time of analysis. Although the correlation between the two architectures confirmed the satisfactory agreement of outputs, this technical note has been thought to provide the reader a fair statement with regard to the strength and drawbacks about combining the two (apparently) competitor devices in a diagnostics field, namely, LFIA and electrochemical strips.

Experimental design for the development of a multiplex antigen lateral flow immunoassay detecting the Southern African Territory (SAT) serotypes of foot-and-mouth disease virus.

Antigenic lateral flow immunoassays (LFIAs) rely on the non-competitive sandwich format, including a detection (labelled) antibody and a capture antibody immobilised onto the analytical membrane. When the same antibody is used for the capture and the detection (single epitope immunoassay), the saturation of analyte epitopes by the probe compromises the capture and lowers the sensitivity. Hence, several factors, including the amount of the probe, the antibody-to-label ratio, and the contact time between the probe and the analyte before reaching the capture antibody, must be adjusted. We explored different designs of experiments (full-factorial, optimal, sub-optimal models) to optimise a multiplex sandwich-type LFIA for the diagnosis and serotyping of two Southern African Territory (SAT) serotypes of the foot-and-mouth disease virus, and to evaluate the reduction of the number of experiments in the development. Both assays employed single epitope sandwich, so most influencing variables on the sensitivity were studied and individuated. We upgraded a previous device increasing the sensitivity by a factor of two and reached the visual limit of detection of 103.7 and 104.0 (TCID/mL) for SAT 1 and SAT 2, respectively. The positioning of the capture region along the LFIA strip was the most influent variable to increase the detectability. Furthermore, we confirmed that the 13-optimal DoE was the most convenient approach for designing the device.

Bacterial ligands as flexible and sensitive detectors in rapid tests for antibodies to SARS-CoV-2.

Lateral flow immunoassay (LFIA) is widely employed as point-of-care tests (POCT) for the diagnosis of infectious diseases. The accuracy of LFIA largely depends on the quality of the immunoreagents used. Typical LFIAs to reveal the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) employ anti-human immunoglobulin (hIG) antibodies and recombinant viral antigens, which usually are unstable and poorly soluble. Broad selective bacterial proteins, such as Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) can be considered alternatives to anti-hIG to increase versatility and sensitivity of serological LFIAs because of their high binding capacity, interspecies reactivity, and robustness. We developed two colorimetric LFA devices including SpA and SpG linked to gold nanoparticles (GNP) as detectors and explored the use of a specific, stable, and soluble immunodominant fraction of the nucleocapsid protein from SARS-CoV-2 as the capturing agent. The optimal amount of SpA-GNP and SpG-GNP conjugates and the protein-to-GNP ratios were defined through a full factorial experimental design to maximize the diagnostic sensitivity of the LFIAs. The new LFA devices were applied to analyze 105 human serum samples (69 positive and 36 negatives according to reference molecular diagnostic methods). The results showed higher sensitivity (89.9%, 95% CI 82.7-97.0) and selectivity (91.7%, 82.6-100) for the SpA-based compared to the SpG-based LFA. In addition, 18 serum samples from cats and dogs living with COVID-19 patients were analyzed and 14 showed detectable levels of anti-SARS-CoV-2 antibodies, thus illustrating the flexibility of the SpA- and SpG-based LFAs.

Ten Years of Lateral Flow Immunoassay Technique Applications: Trends, Challenges and Future Perspectives.

The Lateral Flow Immunoassay (LFIA) is by far one of the most successful analytical platforms to perform the on-site detection of target substances. LFIA can be considered as a sort of lab-in-a-hand and, together with other point-of-need tests, has represented a paradigm shift from sample-to-lab to lab-to-sample aiming to improve decision making and turnaround time. The features of LFIAs made them a very attractive tool in clinical diagnostic where they can improve patient care by enabling more prompt diagnosis and treatment decisions. The rapidity, simplicity, relative cost-effectiveness, and the possibility to be used by nonskilled personnel contributed to the wide acceptance of LFIAs. As a consequence, from the detection of molecules, organisms, and (bio)markers for clinical purposes, the LFIA application has been rapidly extended to other fields, including food and feed safety, veterinary medicine, environmental control, and many others. This review aims to provide readers with a 10-years overview of applications, outlining the trends for the main application fields and the relative compounded annual growth rates. Moreover, future perspectives and challenges are discussed.

Recent Advancements in Enzyme-Based Lateral Flow Immunoassays.

Paper-based lateral-flow immunoassays (LFIAs) have achieved considerable commercial success and their impact in diagnostics is continuously growing. LFIA results are often obtained by visualizing by the naked eye color changes in given areas, providing a qualitative information about the presence/absence of the target analyte in the sample. However, this platform has the potential to provide ultrasensitive quantitative analysis for several applications. Indeed, LFIA is based on well-established immunological techniques, which have known in the last year great advances due to the combination of highly sensitive tracers, innovative signal amplification strategies and last-generation instrumental detectors. All these available progresses can be applied also to the LFIA platform by adapting them to a portable and miniaturized format. This possibility opens countless strategies for definitively turning the LFIA technique into an ultrasensitive quantitative method. Among the different proposals for achieving this goal, the use of enzyme-based immunoassay is very well known and widespread for routine analysis and it can represent a valid approach for improving LFIA performances. Several examples have been recently reported in literature exploiting enzymes properties and features for obtaining significative advances in this field. In this review, we aim to provide a critical overview of the recent progresses in highly sensitive LFIA detection technologies, involving the exploitation of enzyme-based amplification strategies. The features and applications of the technologies, along with future developments and challenges, are also discussed.

Switching from Multiplex to Multimodal Colorimetric Lateral Flow Immunosensor.

Multiplex lateral flow immunoassay (LFIA) is largely used for point-of-care testing to detect different pathogens or biomarkers in a single device. The increasing demand for multitargeting diagnostics requires multi-informative single tests. In this study, we demonstrated three strategies to upgrade standard multiplex LFIA to multimodal capacity. As a proof-of-concept, we applied the strategies to the differential diagnosis of Human Immunodeficiency Virus (HIV) infection, a widespread pathogen, for which conventional multiplex LFIA testing is well-established. In the new two-parameter LFIA (x2LFIA), we exploited color encoding, in which the binding of multiple targets occurs in one reactive band and the color of the probe reveals which one is present in the sample. By combining the sequential alignment of several reactive zones along the membrane of the LFIA strip and gold nanoparticles and gold nanostars for the differential visualization, in this demonstration, the x2LFIA can furnish information on HIV serotype and stage of infection in a single device. Three immunosensors were designed. The use of bioreagents as the capturing ligand anchored onto the membrane or as the detection ligand labelled with gold nanomaterials affected the performance of the x2LFIA. Higher detectability was achieved by the format involving the HIV-specific antigens as capturing agent and labelled secondary bioligands (anti-human immunoglobulins M and protein G) as the probes.

Silver and gold nanoparticles as multi-chromatic lateral flow assay probes for the detection of food allergens.

In this study, we report the simultaneous use of gold and silver nanoparticles to set a multicolor multiplex lateral flow immunoassay (xLFIA). Silver nanoparticles (AgNPs), spherical in shape and characterized by a brilliant yellow color, were obtained by a new viable one-step synthetic protocol. AgNPs were stable over time and acceptably robust to conditions used for fabricating LFIA devices. These AgNPs were employed as a colorimetric probe in combination with two different kinds of gold nanoparticles (AuNPs) to set a visual xLFIA for detecting allergens. Surface plasmon resonance peaks of probes (AgNPs, spherical and desert rose-like AuNPs) were centered at 420, 525, and 620 nm, respectively. Therefore, the xLFIA output was easily interpreted through a "yellow magenta cyan (YMC)" color code. The prospect of the YMC xLFIA was demonstrated by simultaneously detecting three major allergens in bakery products. Antibodies directed towards casein, ovalbumin, and hazelnut allergenic proteins were individually adsorbed onto metal nanoparticles to produce three differently colored specific probes. These were inserted in a LFIA comprising three lines, each responsive for one allergen. The trichromatic xLFIA was able to detect allergenic proteins at levels as low as 0.1 mg/l and enabled the easy identification of the allergens in commercial biscuits based on the color of the probes. Graphical Abstract.

A versatile and sensitive lateral flow immunoassay for the rapid diagnosis of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies, and several domestic and wild mammals act as reservoirs for the infection, so the prompt detection of infected hosts is crucial to preventing and controlling the spread of the disease and its transmission to humans. A rapid and portable tool for VL diagnosis based on the lateral flow immunoassay (LFIA) technology is described herein. The device exploits a highly specific chimeric recombinant antigen as the recognition element for capturing anti-leishmanial antibodies, and protein A labelled with gold nanoparticles as the signal reporter. The LFIA shows excellent diagnostic sensitivity (98.4%), specificity (98.9%), and agreement with serological reference methods for diagnosing canine VL. The long-term stability of the LFIA device was confirmed based on six months of storage at room temperature or 4 °C, and the qualitative response of the device was not affected by limited thermal stress. The use of the broadly specific protein A means that the LFIA can be readily adapted to diagnose VL in dogs (the main reservoir for human infection) and other mammals, thus further assisting efforts to control the spread of VL. Graphical abstract A rapid and portable diagnostic tool for visceral leishmaniasis (VL) based on lateral flow immunoassay (LFIA) technology. The presence of anti-leishmanial antibodies is revealed through the binding of these antibodies to a highly specific chimeric antigen. Employing a broadly specific signal reporter (protein A labelled with gold nanoparticles) enables the LFIA to be easily adapted to diagnose VL in different animals.

A lateral flow immunoassay for straightforward determination of fumonisin mycotoxins based on the quenching of the fluorescence of CdSe/ZnS quantum dots by gold and silver nanoparticles.

A lateral flow immunoassay (LFIA) was developed for the determination of fumonisin mycotoxins. The fluorescence of CdSe/ZnS quantum dots (QDs), observed at excitation/emission wavelengths of 365/525 nm, is suppressed by the addition of silver nanoparticles (SNPs) or gold nanoparticles (GNPs) because SNPs overlap the excitation bands of the QDs, and GNPs overlap the emission bands. The fluorescence of the QDs is recovered upon addition of fumonisins, allowing for the sensitive detection in "positive mode" of the target mycotoxin by monitoring the changes of the QDs fluorescence intensity. The SNPs are found to be the most effective quenchers, while the use of GNPs allows for an efficient recovery of fluorescence and can be employed in the LFIA. The method was successfully applied to the fluorometric determination of fumonisins in spiked maize flour samples. The visual detection limit is at the ng·mL-1 level. This is four times lower compared to the colorimetric LFIA based on the use of the conventional gold NPs. Graphical abstract Schematic of the fluorescence quenching lateral flow immunoassay that uses fluorescent quantum dots (QD) and metal nanoparticles (NP) as the quencher: the binding of NP-labelled antibody to the antigen (purple triangle) modulates QD luminescence at the Test line, allowing for 'positive mode' detection of fumonisins. The NP accumulation at Control line assures validity of the test.

Miniaturized Biosensors to Preserve and Monitor Cultural Heritage: from Medical to Conservation Diagnosis.

The point-of-care testing concept has been exploited to design and develop portable and cheap bioanalytical systems that can be used on-site by conservators. These systems employ lateral flow immunoassays to simultaneously detect two proteins (ovalbumin and collagen) in artworks. For an in-depth study on the application of these portable biosensors, both chemiluminescent and colorimetric detections were developed and compared in terms of sensitivity and feasibility. The chemiluminescent system displayed the best analytical performance (that is, two orders of magnitude lower limits of detection than the colorimetric system). To simplify its use, a disposable cartridge was designed ad hoc for this specific application. These results highlight the enormous potential of these inexpensive, easy-to-use, and minimally invasive diagnostic tools for conservators in the cultural heritage field.

Chemiluminescence lateral flow immunoassay cartridge with integrated amorphous silicon photosensors array for human serum albumin detection in urine samples.

A novel and disposable cartridge for chemiluminescent (CL)-lateral flow immunoassay (LFIA) with integrated amorphous silicon (a-Si:H) photosensors array was developed and applied to quantitatively detect human serum albumin (HSA) in urine samples. The presented analytical method is based on an indirect competitive immunoassay using horseradish peroxidase (HRP) as a tracer, which is detected by adding the luminol/enhancer/hydrogen peroxide CL cocktail. The system comprises an array of a-Si:H photosensors deposited on a glass substrate, on which a PDMS cartridge that houses the LFIA strip and the reagents necessary for the CL immunoassay was optically coupled to obtain an integrated analytical device controlled by a portable read-out electronics. The method is simple and fast with a detection limit of 2.5 mg L-1 for HSA in urine and a dynamic range up to 850 mg L-1, which is suitable for measuring physiological levels of HSA in urine samples and their variation in different diseases (micro- and macroalbuminuria). The use of CL detection allowed accurate and objective analyte quantification in a dynamic range that extends from femtomoles to picomoles. The analytical performances of this integrated device were found to be comparable with those obtained using a charge-coupled device (CCD) as a reference off-chip detector. These results demonstrate that integrating the a-Si:H photosensors array with CL-LFIA technique provides compact, sensitive and low-cost systems for CL-based bioassays with a wide range of applications for in-field and point-of-care bioanalyses. Graphical Abstract A novel integrated portable device was developed for direct quantitative detection of human serum albumin (HSA) in urine samples, exploiting a chemiluminescence lateral flow immunoassay (LFIA). The device comprises a cartridge that holds the LFIA strip and all the reagents necessary for the analysis, an array of amorphous silicon photosensors, and a custom read-out electronics.

A multiplex chemiluminescent biosensor for type B-fumonisins and aflatoxin B1 quantitative detection in maize flour.

A multiplex chemiluminescent biosensor for simple, rapid and ultrasensitive on-site quantification of aflatoxin B1 and type B-fumonisins in maize samples has been developed. The biosensor integrates a multiplex indirect competitive lateral flow immunoassay (LFIA) based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a lensless "contact" imaging configuration. The developed assay requires a simple extraction of the analytes from maize flour samples followed by their detection with a 30 min assay time. The use of chemiluminescence detection allowed accurate and objective analytes quantification, enabling simultaneous detection of type B-fumonisins and aflatoxin B1 down to 6 µg kg(-1) and 1.5 µg kg(-1), respectively, thus fulfilling the standards imposed by the legislation of European Union. Maize flour samples spiked with both analytes were subjected to multiplex analysis obtaining recoveries ranging from 80 to 115% and the coefficient of variation below 20%. Finally, analysis of naturally contaminated maize samples resulted in a good agreement between CL-LFIA and a validated confirmatory HPLC-UV and commercial ELISA kit, obtaining recoveries in the range 88-120%. The proposed CL-LFIA protocol is rapid, inexpensive, easy-to-use, and fit for the purpose of rapid screening of mycotoxins in maize flour.

Mycotoxins-Imprinted Polymers: A State-of-the-Art Review.

Mycotoxins are toxic metabolites of molds which can contaminate food and beverages. Because of their acute and chronic toxicity, they can have harmful effects when ingested or inhaled, posing severe risks to human health. Contemporary analytical methods have the sensitivity required for contamination detection and quantification, but the direct application of these methods on real samples is not straightforward because of matrix complexity, and clean-up and preconcentration steps are needed, more and more requiring the application of highly selective solid-phase extraction materials. Molecularly imprinted polymers (MIPs) are artificial receptors mimicking the natural antibodies that are increasingly being used as a solid phase in extraction methods where selectivity towards target analytes is mandatory. In this review, the state-of-the-art about molecularly imprinted polymers as solid-phase extraction materials in mycotoxin contamination analysis will be discussed, with particular attention paid to the use of mimic molecules in the synthesis of mycotoxin-imprinted materials, to the application of these materials to food real samples, and to the development of advanced extraction methods involving molecular imprinting technology.

The Amount of Cross-Linker Influences Affinity and Selectivity of NanoMIPs Prepared by Solid-Phase Polymerization Synthesis.

The cross-linker methylene-bis-acrylamide is usually present in nanoMIPs obtained by solid-phase polymerization synthesis at 2 mol% concentration, with very few exceptions. Here, we studied the influence of variable amounts of methylene-bis-acrylamide in the range between 0 (no cross-linker) and 50 mol% concentration on the binding properties of rabbit IgG nanoMIPs. The binding parameters were determined by equilibrium binding experiments and the results show that the degree of cross-linking defines three distinct types of nanoMIPs: (i) those with a low degree of cross-linking, including nanoMIPs without cross-linker (0-05 mol%), showing a low binding affinity, high density of binding sites, and low selectivity; (ii) nanoMIPs with a medium degree of cross-linking (1-18 mol%), showing higher binding affinity, low density of binding sites, and high selectivity; (iii) nanoMIPs with a high degree of cross-linking (32-50 mol%), characterized by non-specific nanopolymer-ligand interactions, with low binding affinity, high density of binding sites, and no selectivity. In conclusion, the results are particularly relevant in the synthesis of high-affinity, high-selectivity nanoMIPs as they demonstrate that a significant gain in affinity and selectivity could be achieved with pre-polymerization mixtures containing quantities of cross-linker up to 10-20 mol%, well higher than those normally used in this technique.

A semi-quantitative visual lateral flow immunoassay for SARS-CoV-2 antibody detection for the follow-up of immune response to vaccination or recovery.

The lateral flow immunoassay (LFIA) technique is largely employed for the point-of-care detection of antibodies especially for revealing the immune response in serum. Visual LFIAs usually provide the qualitative yes/no detection of antibodies, while quantification requires some equipment, making the assay more expensive and complicated. To achieve visual semi-quantification, the alignment of several lines (made of the same antigen) along a LFIA strip has been proposed. The numbering of the reacting lines has been used to correlate with the quantity of some biomarkers in serum. Here, we designed the first semiquantitative LFIA for detecting antibodies and applied it to classify the immune response to SARS-CoV-2 raised by vaccination or natural infection. We used a recombinant spike receptor-binding domain (RBD) as the specific capture reagent to draw two test lines. The detection reagent was selected among three possible ligands that are able to bind to anti-spike human antibodies: the same RBD, staphylococcal protein A, and anti-human immunoglobulin G antibodies. The most convenient detector, adsorbed on gold nanoparticles, was chosen based on the highest correlation with an antibody titre of 171 human sera, measured by a reference serological method, and was the RBD (Spearman's rho = 0.84). Incorporated into the semiquantitative LFIA, it confirmed the ability to discriminate high- and low-titre samples and to classify them into two classes (Dunn's test, P < 0.05). The proposed approach enabled the semiquantification of the immune response to SARS-CoV-2 by the unaided eye observation, thus overcoming the requirement of costly and complicated equipment, and represents a general strategy for the development of semiquantitative serological LFIAs.

MIP-based immunoassays: A critical review.

Molecularly imprinted polymers, MIPs, are man-made receptors mimicking the thermodynamic and kinetic binding behaviour of natural antibodies. Therefore, it is not surprising that many researchers have thought about MIPs as artificial receptors in immunoassay-like analytical applications, where the general machinery of the assay is maintained, but the molecular recognition is no longer assured by an antibody but by an artificial receptor. However, the number of papers devoted explicitly to applications of MIPs in the immunoassay field is quite limited if compared to the huge number of papers covering the multifaceted molecular imprinting technology. For this reason, this critical review wants to give a general view of MIP-based immunoassays, trying to highlight the critical points that have so far prevented a wider application of molecular imprinting technology in the immunoassay field and, possibly, try to suggest strategies to overcome them.

Are the NPS commonly used? An extensive investigation in Northern Italy based on hair analysis.

New psychoactive substances (NPS) are present on the Italian illicit markets, but data from the analysis of biological samples to evaluate their real consumption are rare. For this reason, an epidemiological study was carried out by means of a ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of 115 NPS on the keratin matrix. A total of 847 hair samples were collected in 2020 and 2021 and analyzed. The sample donors were in the age range of 18-40 years, from both genders, and were tested either for driving relicensing or for drug withdrawal monitoring. The UPLC-MS-MS system consisted of a Waters ACQUITY UPLC® I-Class, coupled with a Waters XEVO TQ-XS triple quadrupole mass spectrometer. The method was developed and fully validated according to international guidelines. Limits of detection were set as the minimum criterion to identify positive samples. Overall, 56 samples resulted positive for ketamine, 35 for norketamine, 6 for fentanyl, 3 for norfentanyl, 3 for 4-ANPP, 3 for MDMB-4en-PINACA, 2 for N,N-DMT, 2 for 5-chloro AB-PINACA, 1 for α-PHP and 1 for methcathinone. NPS were detected in a small part of samples (8.4%), which seems in contrast with their apparent wide diffusion in Italy, yet it is congruent with similar investigations based on hair analysis. Future studies will be performed to expand the investigated population, especially in terms of age and origin.

Development of molecular and antigenic-based rapid tests for the identification of African swine fever virus in different tissues.

African swine fever (ASF) is a severe haemorrhagic infectious disease affecting suids, thus representing a great economic concern. Considering the importance of the early diagnosis, rapid point of care testing (POCT) for ASF is highly demanded. In this work, we developed two strategies for the rapid onsite diagnosis of ASF, based on Lateral Flow Immunoassay (LFIA) and Recombinase Polymerase Amplification (RPA) techniques. The LFIA was a sandwich-type immunoassay exploiting a monoclonal antibody directed towards the p30 protein of the virus (Mab). The Mab was anchored onto the LFIA membrane to capture the ASFV and was also labelled with gold nanoparticles for staining the antibody-p30 complex. However, the use of the same antibody for capturing and as detector ligand showed a significant competitive effect for antigen binding, so required an experimental design to minimize reciprocal interference and maximize the response. The RPA assay, employing primers to the capsid protein p72 gene and an exonuclease III probe, was performed at 39 °C. The limit of detection of the method was assessed using a plasmid encoding the target gene and resulted in 5 copy/µL. The new LFIA and RPA were applied for ASFV detection in the animal tissues usually analysed by conventional assays (i.e., real-time PCR), such as kidney, spleen, and lymph nodes. A simple and universal virus extraction protocol was applied for sample preparation, followed by DNA extraction and purification for the RPA. The LFIA only required the addition of 3% H2O2 to limit matrix interference and prevent false positive results. The two rapid methods (25 min and 15 min were needed to complete the analysis for RPA and LFIA, respectively) showed high diagnostic specificity (100%) and sensitivity (93% and 87% for LFIA and RPA, respectively) for samples with high viral load (Ct < 27). False negative results were observed for samples with low viral load (Ct > 28) and/or also containing specific antibodies to ASFV, which decreased antigen availability and were indicative of a chronic, poorly transmissible infection. The simple and rapid sample preparation and the diagnostic performance of the LFIA suggested its large practical applicability for POC diagnosis of ASF.

Effect of Surfactants on the Binding Properties of a Molecularly Imprinted Polymer.

In molecularly imprinted polymers, non-specific interactions are generally based on weak forces between the polymer surface and the sample matrix. Thus, additives able to interfere with such interactions should be able to significantly reduce any non-specific binding effect. Surfactants represent an interesting class of substances as they are cheap and easily available. Here, we present a study of the effect of three surfactants (the anionic sodium dodecylsulphate, SDS, the cationic cetyltrimethylammonium bromide (CTAB) and the non-ionic polyoxyethylene-(20)-sorbitan monolaurate Tween 20) on the binding affinity of a 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-imprinted polymer for the template and its analogue 2,4-dichlorophenoxyacetic acid (2,4-D). The experimental results indicate that increasing amounts of surfactant decrease the binding affinity for the ligands strongly for the ionic ones, and more weakly for the non-ionic one. This effect is general, as it occurs for both 2,4,5-T and 2,4-D and for both the imprinted and the not-imprinted polymers. It also proves that the magnitude of this effect mainly depends on the presence or absence of an ionic charge, and that the hydrophobic "tail" of surfactants plays only a minor role.

Rabbit IgG-imprinted nanoMIPs by solid phase synthesis: the effect of cross-linkers on their affinity and selectivity.

Solid phase synthesis (SPS) of molecularly imprinted nanopolymers (nanoMIPs) represents an innovative method to prepare nanomaterials with tailor-made molecular recognition properties towards peptides and proteins. The synthesis of nanoMIPs by SPS usually involves a pre-polymerization formulation, where the cross-linker is invariably N,N'-methylen-bis-acrylamide (BIS). To date, the effect of cross-linkers on the binding properties of nanoMIPs prepared using cross-linkers other than BIS has never been reported. In this work, in order to investigate the effect of different cross-linkers in protein-imprinted nanoMIPs prepared by SPS, alongside BIS we considered other similar cross-linkers: N,N'-ethylene dimethacrylamide (EDAM), N,O-bis-methacryloylethanolamine (NOBE), ethylene glycol dimethacrilate (EDMA) and glycerol dimethacrylate (GDMA), replacing BIS with them in pre-polymerization mixtures. The synthetized nanoMIPs were homogeneous, with a polydispersity index of 0.24-0.30 and a mean diameter of 129-169 nm in water. The binding properties of the nanoMIPs were measured via equilibrium partition experiments with the template, rabbit IgG (RIgG), and the selectivity was evaluated with respect to bovine IgG (BIgG), bovine serum albumin (BSA) and hen egg lysozyme (LZM). The experimental results show that all the cross-linkers, with the exception of EDMA, endowed nanoMIPs with high binding affinities for the template (BIS: 16.0 × 106 mol-1 L, EDAM: 8.8 × 106 mol-1 L, NOBE: 15.8 × 106 mol-1 L, and GDMA: 12.8 × 106 mol-1 L), medium to high imprinting factors (BIS: 12.3, EDAM: 5.5, NOBE: 7.2, and GDMA: 11.6) and good selectivity towards other proteins but markedly dependent on the structure of the cross-linker, confirming the importance of the latter in the SPS of imprinted nanopolymers.