Goat Anti-mouse Immunoglobulin Research Articles

Goat anti-mouse immunoglobulin as “crosslinker” assisted signal tracer assemble with intensive antibody utilization efficiency for sensitive paper-based strip nanobiosensors

Engineered collaborative biochemical techniques and regulated nanomaterials (NMs) offer extraordinary opportunities for improving the analysis performance of lateral flow immunoassay (LFIA). Herein, inspired by the ability of macromolecules (e.g., proteins) to assemble into new functional units and the remarkable optical performance of engineered regulated NMs, goat anti-mouse immunoglobulin (GAMI) serves as the “crosslinker” integrate with gold‑manganese oxide (Au-MnOx) to assemble the “signal tracers (STs)-crosslinker-antibody (mAb)” for elevating the mAb utilization efficiency. Notably, the “STs-crosslinker-mAb” assembly shows ~13.33-folds mAb utilization efficiency enhance, which perfectly response the challenge between limited sensitivity and sufficient signal intensity in competitive-type LFIA. The black color and rough structure of Au-MnOx offer higher colorimetric brightness (~2-folds than AuNPs) and enhanced mAb coupling efficiency (up to 92.47%), which further improves sensitivity under the premise of functional assembly to intensify the competitive immunoreaction. Additionally, the convenient synthesis conditions (~13 min at room temperature) even comparable to direct purchase commercial products indicate that using Au-MnOx undoubtedly increases the cost-effectiveness. Encouragingly, the Au-MnOx-GAMI-mAb based LFIA exhibited high sensitivity (LOD: 0.063 ng mL−1 for clenbuterol (CLE) monitoring) by elevating mAb utilization efficiency with the attendant enhancing immune competition response in a cost-effective manner, which provides an invigorating reference pathway in point-of-care immunoassay.

An automatic and smart platform for rapid detection of cadmium and lead simultaneously in rice using triple-amplified chemiluminescence immunoassay

Rapid detection of trace ions is urgently needed for large-scale screening to ensure food safety. This study developed an innovative and automatic strategy, based on a smart-designed platform for rapid detection of cadmium and lead in rice. As bridge antibody, the antigen was conjugated with goat anti-mouse immunoglobulin G labeled alkaline phosphatase. Meanwhile, a biotin-streptavidin system was introduced to micromagnetic particles, thus providing a triple-amplified chemiluminescence immunoassay with high sensitivity, accuracy and specificity. The limits of detection for cadmium and lead were 0.06 and 1.00 ng mL−1, respectively, within 30 min. The recoveries ranged from 89.81 to 114.92 %, with relative standard deviations less than 9.2 %. The results obtained agreed with those of inductively coupled plasma–mass spectrometry and certified reference materials. Additionally, the auto-operation avoided human errors as well as being convenient, fast, automatic and high-throughput. Therefore, this smart platform can be applied for large-scale Cd2+ and Pb2+ screening.

"Potential Scalpel": A Bioassisted Ultrafast Staining Lateral Flow Immunoassay from De Novo to Results.

It is of great importance to overcome potential incompatibility problems between dyestuffs and antibodies (mAbs) for extensive commercial application of a dyestuff-chemistry-based ultrafast colorimetric lateral flow immunoassay (cLFIA). Herein, inspired by traditional staining technologies, a basic dyestuff gallocyanine (GC)-assisted biogenic "potential scalpel"-based cLFIA (GC-ABPS-based cLFIA) by employing clenbuterol (CLE) as proof-of-concept was proposed to solve a high degree of incompatibility between the same potential dyestuffs and mAbs. Goat antimouse immunoglobulin (Ab2) could serve as the "potential scalpel" to form the positive potential value biomolecular network self-assemblers (BNSA) with anti-CLE mAbs (AbCLE) by noncovalent force. The cLFIA completed the entire detection process from de novo to detection results within 30 min thanks to the easy availability and ideal marking efficiency (≤1 min, saving 0.4-10 h) of GC. Encouragingly, the proposed ultrafast GC-ABPS-based cLFIA has also exhibited high sensitivity (0.411 ng mL-1) and low cost (300 times) compared with other cLFIAs. Also, the feasibility of the proposed cLFIA was demonstrated by detecting CLE in beef, pork ham, and skim milk. Finally, the proposed GC-ABPS-based cLFIA has broadened the application range of dyestuffs and provided an effective reference strategy for the application of dyestuffs in food safety monitoring.

Bioresource-derived tannic acid-supported immuno-network in lateral flow immunoassay for sensitive clenbuterol monitoring

Although lateral flow immunoassay (LFIA) as a point-of-care (POC) diagnostic tool has been widely used because of numerous merits, it remains challenging to realize higher sensitivity, easier labeling, and fewer antibodies consumption. Herein, a bioresource-derived tannic acid (TA)-supported immuno-network based LFIA for clenbuterol (CLE) monitoring was proposed by employing poly TA nanospheres (PTAN) as the new tracer. Attributing to the effective protein-enrichment ability of TA, plenty of goat anti-mouse immunoglobulins (GAMI) were first absorbed on the surface of PTAN and then monoclonal antibodies (AbCLE) were added to form immuno-network. Compared with directly coupling antibodies, the utilization efficiency of AbCLE was significantly improved. Thus, the detection sensitivity (take the cut-off value as an example) was enhanced 5-fold and 10-fold at least compared with PTAN-AbCLE and colloidal gold-based LFIA, respectively. Additionally, the proposed method was successfully verified in beef and pork liver and provided an effective strategy for food safety monitoring.

Hybridoma Screening by Antigen Capture: Reverse Dot Blot.

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then "captured" on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).

The early detection of type 1 diabetes mellitus and latent autoimmune diabetes in adults (LADA) through rapid test reverse-flow immunochromatography for glutamic acid decarboxylase 65 kDa (GAD65)

BackgroundDiabetes mellitus (DM) is a chronic and costly disease that has become a primary concern worldwide. Type 1 diabetes mellitus is categorized as an autoimmune disease, which results in islet cell apoptosis and insulin-dependent. GAD65 is known as a potential marker of impaired pancreatic β cell function that appears in the initial phase of type 1 DM and latent autoimmune diabetes in adults (LADA). This study aimed to develop a novel rapid test of anti-GAD65 autoantibodies in human serum samples. MethodsWe have developed a rapid test for anti-GAD65 autoantibodies in this assay based on the reverse-flow immunochromatography method. Human recombinant-protein antigen for GAD65 was attached as the control line over the nitrocellulose membrane. On the other side, the goat anti-mouse immunoglobulin G (IgG) was coated on the same membrane as a control line. The positive result for GAD65 was confirmed by a colloidal gold signal on the strip. Our novel assay analyzed 276 healthy subjects and 51 type 1 diabetes individuals serum samples from several ethnicities in Indonesia for this study. ResultsAmong the 276 healthy samples, 225 samples were identified as positive for anti-GAD65 autoantibodies, while 51 samples were negative. Interestingly, the positive results for anti-GAD65 autoantibodies were linear to the decreasing of high-density lipoprotein (HDL) levels and inversely associated with triglyceride levels. A significant correlation with age was observed in all groups. The sensitivity and specificity test proved that this kit has higher accuracy (AUC value = 0.960). ConclusionThe significant advantages of our rapid test for anti-GAD65 autoantibodies provide higher sensitivity, specificity, and stability compared to previous commercial kits. Therefore, it could be proposed as the future clinical diagnostic kit for patient management of type 1 DM.

Novel dual immunochromatographic test strip based on double antibodies and biotin-streptavidin system for simultaneous sensitive detection of aflatoxin M1 and ochratoxin A in milk

The coexistence of mycotoxins in agricultural products poses a serious threat to food safety. This study developed a dual immunochromatographic test strips (DICTS) method based on double antibodies labeled with time-resolved fluorescent microspheres (TRFM) to realize simultaneous rapid detection of aflatoxin M1 (AFM1) and ochratoxin A (OTA) in milk. As bridge antibody, the polyclonal antibody (pAb) was first conjugated with the TRFM and then with the monoclonal antibody (mAb). Meanwhile, a biotin-streptavidin system was introduced to replace the traditional goat anti-mouse Immunoglobulin G, thus providing a stable signal on the control line. After optimization, the detection limit of AFM1 and OTA in milk was respectively 0.018 and 0.036 ng/mL. The recoveries of intraassay and interassay experiments ranged from 89.65% to 103.99%. The accuracy, repeatability, and specificity of the developed TRFM-DICTS were estimated. The results of TRFM-DICTS showed a high consistency with those of the ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Development of a multi-channel magnetic bead micro-probe assay for high-throughput detection of zearalenone in edible and medicinal Coix seed

A multi-channel magnetic bead micro-probes assay (MBPA) based on indirect competitive principle was developed for high-throughput detection of zearalenone (ZEA) in edible and medicinal Coix seed. This strategy introduced magnetic beads as the carriers, the specific primary antibodies as the capture probes for targets and the secondary antibodies functionalized goat anti-mouse immunoglobulin G labeled fluorescein isothiocyanate as the fluorescence signal probes. Through the competitive reaction of ZEA in Coix seed samples and that covalently coupled on the surface of MBs with their specific antibodies, as well as fast magnetic separation and sensitive fluorescence detection, the developed MBPA strategy allowed low limit of detection (2.03 ng/mL) with broad dynamic range (2.03–440.67 ng/mL), as well as excellent accuracy with the average recovery rate of 96.39% and relative standard deviation (RSD) of 5.48% for ZEA. 36 samples could realize simultaneous analysis in one operation within less than 20 min only needing 50 μL of solution and 30 s of sampling, avoiding large consumption of time and organic solvents. Multiple centrifugation and cleanup steps were omitted because of magnetic separation, avoiding the loss of targets. Diverse capture and fluorescent probes can be randomly bound onto the surface of MBs, making the MBPA strategy a promising tool for on-site high-throughput monitoring of various trace hazard factors in food safety, and environmental monitoring.

Resolved Infrared Spectroscopy of Aqueous Molecules Employing Tunable Graphene Plasmons in an Otto Prism.

Real-time and in situ detection of aqueous solution is essential for bioanalysis and chemical reactions. However, it is extremely challenging for infrared microscopic measurement because of the large background of water absorption. Here, we proposed a wideband-tunable graphene plasmonic infrared biosensor to detect biomolecules in an aqueous environment, employing attenuated total reflection in an Otto prism configuration and tightly confined plasmons in graphene nanoribbons. Benefiting from the graphene plasmonic electric field enhancement, such a biosensor is able to identify the molecular chemical fingerprints without the interference of water absorption. As a proof of concept, the recombinant protein AG and goat anti-mouse immunoglobulin G (IgG) are used as the sensing analytes, of which the vibrational modes (1669 and 1532 cm-1) are very close to the OH-bending mode of water (1640 cm-1). Simulation results show that the fingerprints of protein molecules in the water environment can be selectively enhanced. Therefore, the water absorption is successfully suppressed so that two protein modes can be resolved by sweeping graphene Fermi energy in a wide waveband. By further optimizing the incident angle and graphene mobility to improve the mode energy of graphene plasmons, maximum enhancement factors of 112 and 130 can be achieved for amide I and II bands. Our work provides an effective approach for the highly sensitive and selective in situ identification of aqueous-phase molecular fingerprints in fields of healthcare, food safety, and biochemical sensing.

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease.

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VPAHPND ), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15min. The sensitivity of the kit is approximately 6.25µg/ml of toxin, which was equivalent to the toxin produced by approximately 107 cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VPAHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.

Cord-Based Microfluidic Chips as A Platform for ELISA and Glucose Assays.

This paper describes the development and application of microfluidic cord-based analytical devices (µCADs) in two enzyme-linked immunosorbent assays (ELISAs) and glucose assay. In this study, biotinylated goat anti-mouse immunoglobulin (IgG) antibody, rabbit IgG antibody, and glucose are quantitatively detected. In the ELISA systems, the antibody is spotted on the cord at the detection site and a series of washes, followed by streptavidin-alkaline phosphatase (Strep-ALP) or alkaline phosphatase (ALP)-conjugated secondary antibody and colorimetric substrate, completing the experiment. The devices are subsequently scanned and analyzed yielding a correlation between inverse yellow or inverse blue intensity and antibody concentration. For the first ELISA, a linear range of detection was observed at lower concentrations (2.50 × 10−4–1.75 × 10−3 mg/mL) of Strep-ALP with saturation of the enzyme achieved at higher concentrations (>2.50 × 10−4). For the second ELISA, the L50 was demonstrated to be 167.6 fmol/zone. The glucose assay consisted of spotting increasing concentrations of glucose on the analysis sites and transporting, via capillary action, a solution containing glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) to the detection sites realizing a yellow-brown color indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed to show the correlation between yellow inverse intensity and glucose. Glucose in artificial urine showed good correlation using the devices.

Получение иммунореагентов для серологического определения возбудителей дитиленхозов сельскохозяйственных и декоративных культур

The purpose of the research is obtaining immuno reagents for determination of two types of parasitic nematodes Ditylenchus destructor and D. dipsaci (antigens and antiserums for them). Materials and methods. Three immunizations of laboratory animals (white scrub mice) have been carried out at a dose of 20 mcg of the target protein per animal at 7–10 day intervals. The efficient titer of the antiserums obtained was described by the Dot-ELISA method using secondary goat anti-mouse immunoglobulins conjugated with alkaline phosphatase. For measuring a specificity of the obtained immuno reagents, an ELISA plate in an indirect format was used. Results and discussion. The antiserums obtained have a specificity for two types of nematodes D. destructor and D. dipsaci, and for one non-parasitic nematode Rhabditis sp. The specificity of the obtained antiserums ranges from 82 to 97%. The sensitivity of antiserums to homogenates of stem nematodes D. destructor and D. dipsaci of plants at the antiserum dilution 1 : 3000 is 1 ng and 50 ng accordingly.

Cytometric Microbead Magnetic Suspension Array for High-Throughput Ultrasensitive Detection of Aflatoxin B1.

High-throughput and low-cost detection of mycotoxins in complex matrices is becoming increasingly urgent but it is still challenging to perform ultrasensitive analyses. Here we report a green and practical cytometric microbead magnetic suspension array (CBMSA) strategy for rapid and economical detection of aflatoxin B1 (AFB1) in multiple batches of lotus seed samples. The protocol included (1) fabrication of suspension array chips by immobilizing biotin-modified bovine serum albumin-AFB1 (antigen) onto the surface of streptavidin-coated magnetic microbeads in a multiwell array, (2) indirect immunocompetition of antigen and target of AFB1 in lotus seed samples with the specific antibodies, (3) rapid magnetic separation regardless of complex pretreatment steps, and (4) ultrasensitive fluorescence detection of fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G (FITC-IgG) probes. After systematic optimization of some crucial parameters, the developed CBMSA assay allowed for ultrasensitive detection of AFB1 with limit of detection as low as 7.8125 pg·kg-1. For high-throughput analysis, the CBMSA technique was capable of on-site co-instantaneous detection of 50-100 samples in one operation within 30 s, only needing a small amount (50 μL) of solution, which is much cheaper, greener, and more user-friendly than conventional techniques. Moreover, CBMSA with magnetic separation is free of multiple centrifugation and cleanup steps to avoid unpredictable loss of targets. Since various capture and fluorescent probes can be randomly constructed and bound onto the surface of magnetic microbeads to establish an ultrasensitive detection system, the CBMSA technique is very promising for more trace analytes in complex matrices and for broad point-of-need applications, such as drug screening and real-time high-throughput analysis.

Eu3+ -labeled IgG-based time-resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1 in feed.

Aflatoxin B1 (AFB1 ) is a kind of toxic and carcinogenic mycotoxin. A time-resolved fluoroimmunoassay (TRFIA) was established for quantitative detection of AFB1 in feed using Eu3+ -labeled IgG as tracer. Monoclonal antibody (McAb) against AFB1 (9B11-D7) was prepared through immunization and cell fusion and was identified as high affinity, specificity and sensibility by enzyme-linked immunosorbent assay (ELISA). The 50% inhibition value (IC50 ) was 0.81 ng mL-1 , the limit of detection (LOD) was 0.10 ng mL-1 and detection range was 0.10-3.94 ng mL-1 . Goat anti-mouse immunoglobulin G (IgG) was modified by Eu3+ -DATT, generating Eu3+ -labeled IgG. Under optimal assay conditions, TRFIA was shown to be highly sensitive and specific in detection of AFB1 . The IC50 and LOD were 94.73 pg mL-1 and 3.55 pg mL-1 , respectively, and detection range was 3.55-1.11 × 103 pg mL-1 . Cross-reactivity with AFM1 , AFB2 , AFG1 and AFG2 was 31.26%, 37.6%, 127.46% and 35.74%, respectively, but zero with other analogues. In determination of AFB1 spiked in feed sample, TRFIA showed high accuracy and precision. The average recoveries ranged from 93.71% to 97.80%, and coefficient of variation was 1.25-3.73%. Good correlation between TRFIA and HPLC was demonstrated for determination of AFB1 in feeds, confirming the reliability of the developed method. The developed TRFIA exhibited good potential for employment in the ultrasensitive detection of AFB1 in feed and could be used to determine total aflatoxins. © 2017 Society of Chemical Industry.

Magnetic microspheres-based cytometric bead array assay for highly sensitive detection of ochratoxin A

Accurate and sensitive quantification of a specific class of mycotoxins at trace levels in complex matrices with greener approaches is of significant importance. In this study, a green and economical protocol of magnetic microspheres-based cytometric bead array (CBA) assay on indirect competitive principle was developed for sensitive and rapid detection of ochratoxin A (OTA) in malts with a small number of standard and sample solutions. The protocol included the competition of OTA in malt samples and that covalently coupled on the surface of microspheres with its monoclonal antibodies, the separation and aggregation of the magnetic microspheres, and the fluorescence detection of fluorescein isothiocyanate labeled goat anti-mouse immunoglobulin G probes. The magnetic microspheres-based CBA assay allowed for ultralow limit of detection (0.025μgkg−1) for OTA and showed higher sensitivity compared with the common polystyrene beads-based CBA method. This is the first report on the magnetic microspheres-based CBA assay by using a simple and easy-to-operate magnetic separator for highly sensitive and rapid detection of OTA in complex malt samples. By consuming less solvent, time and cost, as well as fewer standard and samples, the developed green protocol expressed high potential for one-site real-time detection of trace components in complex matrices.

High sensitivity immunochromatographic strip test (ICP11 strip test) for white spot syndrome virus detection using monoclonal antibodies specific to ICP11 non-structural protein

Two monoclonal antibodies (MAbs) specific to different epitopes on ICP11, a non-structural protein of white spot syndrome virus (WSSV), were employed to develop a highly sensitive immunochromatographic strip test (ICP11 strip test). One MAb (WI-11) was conjugated to colloidal gold to bind to ICP11 and another MAb (WI-16) was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. A downstream control line (C) of goat anti-mouse immunoglobulin G (GAM) antibody was used to capture the free colloidal gold conjugated MAb to validate the test performance. Homogenate of shrimp sample in application buffer could be applied directly to the application well and the test result was obtained within 15min. The sensitivity of the kit is 40 times more sensitive than the detection limit of previously reported strip test using MAbs specific to VP28, and approximately 400 times more sensitive than that of Shrimple®, but approximately 50 times less sensitive than that of one-step PCR. For experimentally WSSV-infected shrimps, the ICP11 strip could detect WSSV as early as 12–18h post infection. Due to its high specificity, simplicity and no requirement on sophisticated equipment or specialized skills, the strip test could be adopted to surveillance of early WSSV infection at the pond site.

Portable detection of clenbuterol using a smartphone-based electrochemical biosensor with electric field-driven acceleration

We have developed a fast, highly sensitive and low-cost biosensing system for the detection of clenbuterol (CLB), using a homemade mobile electrochemical device with an electric field-driven acceleration strategy. This system consists of an embedded circuit in smartphone for signal processing and a screen-printed carbon electrode (SPCE) modified with multi-walled carbon nanotubes (MWNTs) and goat anti mouse-immunoglobulin G (IgG) sensing layer (MWNTs-I-layer). CLB monoclonal antibody was assembled through its binding to the surface-confined antibody. Such modified electrodes were used for rapid and sensitive amperometric immunosensing detection of CLB. Horseradish peroxidase-coupled CLB (CLB–HRP) competed with free CLB in the samples to bind the monoclonal antibody. By using this mobile system, we could detect CLB ranging from 0.3ng⋅mL−1 to 100ng⋅mL−1 with the detection limit of 0.076ng⋅mL−1. The whole competitive-type detection process was finished within 6min. We expect this device can meet the requirements for field detection of various food security-related species.

Synthesis and characterization of C@CdS dots in aqueous solution and their application in labeling human gastric carcinoma cells

Colloidal carbon spheres coated with cadmium sulfide nanoparticle quantum dots (C@CdS dots) with the particle size smaller than 50 nm were synthesized by an aqueous approach. The effects of different reaction times, temperatures, and pH values were carefully investigated to optimize the synthesis conditions. The as-prepared C@CdS dots were linked with mouse anti-human carcinoembryonic antigen antibody and goat anti-mouse immunoglobulin (IgG) to directly and indirectly label fixed human gastric carcinoma cells, respectively. The cytotoxicity of the C@CdS dots was also tested using the human gastric carcinoma cells. No apparent cytotoxicity was observed, which suggested the potential application of the as-prepared C@CdS dots in bioimaging.

Application of an OLED integrated with BEF and giant birefringent optical (GBO) film in a SPR biosensor

This study aims to improve the signal sensitivity of a portable surface plasmon resonance (SPR) sensor system. An organic light emitting diode (OLED) was integrated with a brightness enhancement film (BEF) and a giant birefringent optical (GBO) film as a light source to construct the SPR based on an OLED–BEF–GBO film. A method for calculating the SPR signal by summing the differences in optical intensity at two different wavelengths was used to minimize the effect of the noise error that particularly occurs in a portable SPR device. The experimental results indicated that the limit of detection (LOD) of the SPR using an Au sensing layer has an effective refractive index change of 7.8×10−6RIU. Furthermore, for the SPR using Au/Ag sensing metal layers, an LOD of 3.2×10−6RIU was achieved due to the improved sensitivity and resolution value compared with the use of an Au sensing layer. The use of the SPR device in a bio-affinity assay to test the interaction between goat anti-mouse immunoglobulin G (IgG) and mouse IgG protein achieved a LOD of approximately 40.6pg/mL.

Enhanced emission of fluorophores on shrink-induced wrinkled composite structures.

We introduce a manufacturable and scalable method for creating tunable wrinkled ferromagnetic-metallic structures to enhance fluorescence signals. Thin layers of nickel (Ni) and gold (Au) were deposited onto a pre-stressed thermoplastic (shrink wrap film) polymer. Heating briefly forced the metal films to buckle when the thermoplastic retracted, resulting in multi-scale composite 'wrinkles'. This is the first demonstration of leveraging the plasmons in such hybrid nanostructures by metal enhanced fluorescence (MEF) in the near-infrared wavelengths. We observed more than three orders of magnitude enhancement in the fluorescence signal of a single molecule of goat anti-mouse immunoglobulin G (IgG) antibody conjugated to fluorescein isothiocyanate, FITC, (FITC-IgG) by two-photon excitation with these structures. These large enhancements in the fluorescence signal at the nanoscale gaps between the composite wrinkles corresponded to shortened lifetimes due to localized surface plasmons. To characterize these structures, we combined fluctuation correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), and two-photon microscopy to spatially and temporally map the hot spots with high resolution.