Versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels

A versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels has been developed for sensitive protein detection. This sandwich-type sensor is fabricated on an indium tin oxide chip covered with a well-ordered gold nanoparticle monolayer. Gel imaging systems were successfully introduced to develop a novel high-efficient optical immunoassay, which could perform simultaneous detection for the samples with a series of different concentrations of a target analyte. The linear range of this assay was between 0.1 and 500 ng/mL, and the assay sensitivity could be further increased to 0.005 ng/mL with the linear range from 0.005 to 100 ng/mL by stripping voltammetric analysis. The immunosensor showed good precision, high sensitivity, acceptable stability, and reproducibility and could be used for the detection of real sample with consistent results in comparison with those obtained by the ELISA method.     

In situ amplified electrochemical immunoassay for carcinoembryonic antigen using horseradish peroxidase-encapsulated nanogold hollow microspheres as labels

Methods based on sandwich-type electrochemical enzyme immunoassay protocol have been extensively developed for the detection of biomolecules, but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. In this study, we initially synthesized specially horseradish peroxidase-encapsulated nanogold hollow microspheres (HRP-GHS), and then the prepared HRP-GHS was conjugated to the secondary carcinoembryonic antibody (HRP-GHS- anti-CEA). Carcinoembryonic antigen (CEA), as a model protein, was monitored by using the electrochemical sandwich-type enzyme immunoassay format. Under optimized conditions, the linear range of the immunoassay by using single HRP-labeled anti-CEA (HRP- anti-CEA) as secondary antibodies is 2.5-120 ng/mL with a detection limit of 1.5 ng/mL CEA, while the assay sensitivity by using HRP-GHS- anti-CEA as secondary antibodies is further increased from 0.01 to 200 ng/mL with a lower detection limit of 1.5 pg/mL CEA. The intra- and interassay reproducibility is acceptable. The CEA concentrations of the clinical serum specimens assayed by the developed immunoassay show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay. This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays.     

Enzyme-amplified aequorin-based bioluminometric hybridization assays

The sensitivity of aequorin-based bioluminometric hybridization assays was enhanced by introducing, enzymically, multiple aequorin labels per DNA hybrid. The target DNA was hybridized in microtiter wells with an immobilized capture probe and a digoxigenin-labeled detection probe. The hybrids were reacted with an anti-digoxigenin antibody conjugated to horseradish peroxidase. Peroxidase catalyzed the oxidation of digoxigenin-tyramine by hydrogen peroxide, resulting in the attachment of multiple digoxigenin moieties to the solid phase. Aequorin-labeled anti-digoxigenin antibody was then allowed to bind to the immobilized digoxigenins. The bound aequorin was determined by its characteristic Ca2+-triggered bioluminescence. As low as 20 fmol/L (1 amol/ well) target DNA was detected with a signal-to-background ratio of 2.7. A hybridization assay that used only aequorin-labeled anti-digoxigenin antibody without the peroxidase amplification step gave a signal-to-background ratio of 2 for 160 fmol/L target DNA. The signal enhancement of the amplified assay was in the range of 14-38 times. The analytical range of the amplified assay extended up to 2600 fmol/L. The CVs were in the range of 5.5-7.3%.     

Enzyme-amplified electrochemical detection of DNA using electrocatalysis of ferrocenyl-tethered dendrimer

We have developed a sandwich-type enzyme-linked DNA sensor as a new electrochemical method to detect DNA hybridization. A partially ferrocenyl-tethered poly(amidoamine) dendrimer (Fc-D) was used as an electrocatalyst to enhance the electronic signals of DNA detection as well as a building block to immobilize capture probes. Fc-D was immobilized on a carboxylic acid-terminated self-assembled monolayer (SAM) by covalent coupling of unreacted amine in Fc-D to the acid. Thiolated capture probe was attached to the remaining amine groups of Fc-D on the SAM via a bifunctional linker. The target DNA was hybridized with the capture probe, and an extension in the DNA of the target was then hybridized with a biotinylated detection probe. Avidin-conjugated alkaline phosphatase was bound to the detection probe and allowed to generate the electroactive label, p-aminophenol, from p-aminophenyl phosphate enzymatically. p-Aminophenol diffuses into the Fc-D layer and is then electrocatalytically oxidized by the electronic mediation of the immobilized Fc-D, which leads to a great enhancement in signal. Consequently, the amount of hybridized target can be estimated using the intensity of electrocatalytic current. This DNA sensor exhibits a detection limit of 20 fmol. Our method was also successfully applied to the sequence-selective discrimination between perfectly matched and single-base mismatched target oligonucleotides.     

Sensitive amperometric immunosensing using polypyrrolepropylic acid films for biomolecule immobilization

An electrochemical immunosensor was constructed using an electropolymerized pyrrolepropylic acid (PPA) film with high porosity and hydrophilicity. A high density of carboxyl groups of PPA was used to covalently attach protein probes, leading to significantly improved detection sensitivity compared with conventional entrapment methods. As a model, anti-mouse IgG was covalently immobilized or entrapped in the PPA film and used in a sandwich-type alkaline phosphatase-catalyzing amperometric immunoassay with p-aminophenyl phosphate as the substrate. With covalent binding, the detection limit for IgG in PBS buffer, pH 7.4, was 100 pg/mL with a dynamic range of 5 orders of magnitude. The covalent bonding mode in the carbonate-bicarbonate buffer, pH 9.6, further brought down the detection limit to 20 pg/mL with remarkable selectivity.     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles

A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.     

Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for Cryptosporidium parvum hsp70 mRNA

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Cryptosporidium parvum in water has been developed. The target molecule was a 121-nucleotide sequence from the C. parvum heat shock protein 70 (hsp70 mRNA from U71181 gene). This analyte offers the possibility of distinguishing dead from live oocysts. The assay involves covalent attachment of a primary DNA probe via its 5'-amine-terminus to self-assembled monolayers of mercaptoundecanoic acid to a gold surface. The primary DNA probe was used to capture the target (sequence 1039-1082 of U71181 gene for the mRNA), by hybridization to a 20-base complementary sequence on the target (at sequence 1063-1082). A secondary DNA probe labeled with alkaline phosphatase (AP) was then hybridized to base sequence 1039-1062 on the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution, in which gold-coated silicon wafer modified with the complete assembly of the assay components was incubated, is linear with concentration of the target (5-50 microg/mL, where P1 and P2-AP concentrations are 50 microg/mL). A detection limit of 2 microg/mL (or 146 nM) of the DNA target was obtained. Cross-reactivity tests showed high selectivity for heat-shocked C. parvum. No signal was obtained for either the synthetic DNA for hsp70 of Campylobacter lari, Escherichia coli, Giardia lamblia, Salmonella typhimurium, and Listeria monocytogenes or for the products of heat-shocked whole organisms of E. coli, G. lamblia, Staphylococcus aureus, and Cryptosporidium muris.     

Silver nanoparticle-based ultrasensitive chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which the DNA targets are first hybridized to the captured oligonucleotide probes immobilized on polystyrene microwells and then the silver nanoparticles modified with alkylthiol-capped oligonucleotides are used as probes to monitor the presence of the specific target DNA. After being anchored on the hybrids, silver nanoparticles are dissolved to Ag+ in HNO3 solution and sensitively determined by a coupling CL reaction system (Ag+-Mn2+-K2S2O8-H3PO4-luminol). The combination of the remarkable sensitivity of the CL method with the large number of Ag+ released from each hybrid allows the detection of specific sequence DNA targets at levels as low as 5 fM. The sensitivity increases 6 orders of magnitude greater than that of the gold nanoparticle-based colorimetric method and is comparable to that of surface-enhanced Raman spectroscopy, which is one of the most sensitive detection approaches available to the nanoparticle-based detection for DNA hybridization. Moreover, the perfectly complementary DNA targets and the single-base mismatched DNA strands can be evidently differentiated through controlling the temperature, which indicates that the proposed CL assay offers great promise for single-nucleotide polymorphism analysis.     

Small-volume detection of Plasmodium falciparum CSP gene using a 50-microm-diameter cavity with self-contained electrochemistry

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1(0)P, which was of two different sequences we call 1(0)P(a) and 1(0)P(b)) was used to capture the target (T, which was of two different sequences, T1 sequence 481-590 and T2 sequence 472-590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1(0)P(a), 47 bases were complementary to T1 and T2 at 543-590, while on 1(0)P(b), 35 bases were complementary to T1 and T2 at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2(0)P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P1 (P1a and P1b) and P2-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.     

A nucleic acid biosensor for gene expression analysis in nanograms of mRNA

An ultrasensitive nucleic acid biosensor for direct detection of genes in mRNA extracted from animal tissues is described. It is based on amperometric detection of a target gene by forming an mRNA/redox polymer bilayer on a gold electrode. The mRNA was directly labeled with cisplatin-biotin conjugates through coordinative bonds with purine bases in the mRNA molecules. A subsequent binding of glucose oxidase-avidin conjugates to the labeled mRNA and the introduction of a poly(vinylimidazole-co-acrylamide) partially imidazole-complexed with [Os(bpy)(2)(im)] (bpy = 2,2'-bipyridine, im = imidazole) redox polymer overcoating to the electrode allowed for electrochemical detection of the oxidation current of glucose in solution. Depending on individual genes, detection limits of subfemtograms were achieved. As compared to a sandwich-type assay, the sensitivity was improved by as much as 25-fold through the incorporation of multiple enzyme labels to the mRNA molecules. Less than 2-fold gene expression difference was unambiguously differentiated in as little as 5.0 ng of mRNA. With the greatly improved sensitivity, at least 1000-fold more sensitive than fluorescence-based techniques, the amount of mRNA needed in the assay was cut down from microgram to nanogram levels.     

Detection of microRNAs using electrocatalytic nanoparticle tags

An ultrasensitive microRNA (miRNA) assay employing electrocatalytic nanoparticle tags to meet the need of miRNA expression analysis is described in this report. The assay utilizes an indium tin oxide electrode on which oligonucleotide capture probes are immobilized. After hybridization with periodate-treated miRNA, the nanoparticle tags, isoniazid-capped OsO2 nanoparticles, are brought to the electrode through a condensation reaction to chemically amplify the signal. The resulting electrode exhibits electrocatalytic activity toward the oxidation of hydrazine at -0.10 V, reducing the oxidation overpotential by as much as 900 mV. The effect of experimental variables on the amperometric response is investigated and optimized. A detection limit of 80 fmol/L in 2.5-microL droplets and a linear current-concentration relationship up to 200 pmol/L are obtained following a 60-min hybridization. Successful attempts are made in miRNA expression analysis of HeLa cells.     

Gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus DNA using disposable microband electrodes

An electrochemical DNA detection method has been developed for the sensitive quantification of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). The assay relies on (i) the hybridization of the single-stranded target HCMV DNA with an oligonucleotide-modified Au nanoparticle probe, (ii) followed by the release of the gold metal atoms anchored on the hybrids by oxidative metal dissolution, and (iii) the indirect determination of the solubilized AuIII ions by anodic stripping voltammetry at a sandwich-type screen-printed microband electrode (SPMBE). Due to the enhancement of the AuIII mass transfer by nonlinear diffusion during the electrodeposition time, the SPMBE allows the sensitive determination of AuIII in a small volume of quiescent solution. The combination of the sensitive AuIII determination at a SPMBE with the large number of AuIII released from each gold nanoparticle probe allows detection of as low as 5 pM amplified HCMV DNA fragment.     

Biosensor for dengue virus detection: sensitive, rapid, and serotype specific

A serotype-specific RNA biosensor was developed for the rapid detection of Dengue virus (serotypes 1-4) in blood samples. After RNA amplification, the biosensor allows the rapid detection of Dengue virus RNA in only 15 min. In addition, the biosensor is portable, inexpensive, and very easy to use, making it an ideal detection system for point-of-care and field applications. The biosensor is coupled to the isothermal nucleic acid sequence-based amplification (NASBA) technique with which small amounts of virus RNA are amplified using a simple water bath. During the NASBA reaction, a generic sequence is attached to all RNA molecules as described earlier (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). It has been shown earlier that Dengue virus can be detected specifically using two DNA probes: a first probe hybridized with the attached generic sequence and, therefore, bound to every amplified RNA molecule; and a second probe either bound to all four Dengue virus serotypes or chosen to be specific for only one serotype. These probes were utilized in the biosensor described in this publication. For a generic Dengue virus biosensor, the second probe is complementary to a conserved region found in all Dengue serotypes. For identification of the individual Dengue virus serotypes, four serotype-specific probes were developed (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtiff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The generic DNA probe (reporter probe) is coupled to the outside of dye-encapsulating liposomes. The conserved or Dengue serotype specific probes (capture probes) are immobilized on a polyethersulfone membrane strip. Liposomes are mixed with amplified target sequence and are then applied to the membrane. The mixture is allowed to migrate along the test strip, and the liposome-target sequence complexes are immobilized in the capture zone via hybridization of the capture probe with target sequence. The amount of liposomes present in the immobilized complex is directly proportional to the amount of target sequence present in the sample and can be quantified using a portable reflectometer. The different biosensor components have been optimized with respect to sensitivity and, foremost, specificity toward the different serotypes. An excellent correlation to a laboratory-based detection system was demonstrated. Finally, the assay was tested using a limited number of clinical human serum samples. Although Dengue serotypes 1, 2 and 4 were identified correctly, serotype 3 displayed low cross-reactivity with biosensors designed for detection of serotypes 1 and 4.     

Signal amplification of graphene oxide combining with restriction endonuclease for site-specific determination of DNA methylation and assay of methyltransferase activity

Site-specific identification of DNA methylation and assay of MTase activity are important in determining specific cancer types, providing insights into the mechanism of gene repression, and developing novel drugs to treat methylation-related diseases. This work reports an electrochemical method for gene-specific methylation detection and MTase activity assay using HpaII endonuclease to improve selectivity and employing signal amplification of graphene oxide (GO) to enhance the assay sensitivity. The method was developed by designing a probe DNA, which was immobilized on electrode surface, to hybridize with target DNA (one 137 mer DNA from exon 8 promoter region of the Homo sapiens p53 gene, was extracted from HCT116 cells). The assay is based on the electrochemical responses of the reporter (thionine), which was conjugated to 3'-terminus of the probe DNA via GO, after the DNA hybrid was methylated (under catalysis of M.SssI MTase) and cleaved by HpaII endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence of 5'-CCGG-3' and catalyzing cleavage between the cytosines). This model can determine DNA methylation at the site of CpG and has an ability to discriminate the target DNA sequence from even single-base mismatched sequence. The electrochemical signal has a linear relationship with M.SssI activities ranging from 0.1 to 450 U/mL with a detection limit of ～(0.05 ± 0.02) U/mL at a signal/noise of 3. The advantages of this assay are ease of performance having a good specificity and selectivity. In addition, we also demonstrate the method can be used for rapid evaluation and screening of the inhibitors of MTase and has a potential application in discovery of new anticancer drugs.     

Nanotube-based colorimetric probe for ultrasensitive detection of ataxia telangiectasia mutated protein

We have developed a nanotube-based colorimetric probe using multiwalled carbon nanotubes (MWNTs), anti-immunoglobulin G (anti-IgG), and horseradish peroxidase (HRP). The probe was used as an alternative to conventional colorimetric conjugates to obtain amplified signals in a sandwich-type immunoassay for ataxia telangiectasia mutated (ATM), a potential biomarker for radiation doses and cancers. Results show that the MWNT-based probe colorimetry was 5000 times more sensitive than a conventional ELISA, while its concentration range was 10,000 times wider than that of the latter. Its limit of detection (LOD) was 0.2 fg/mL (54 aM, ~32 molecules in 1 μL samples). Control experiments showed that detection of ATM molecules at the picogram-level could still be achieved in samples that contained protein makers present at more than 100 times the ATM concentration, demonstrating the high specificity of the technique. The MWNT-based probe also has the potential to become a universal probe for colorimetric assays of most protein markers because it can recognize the associated rabbit polyclonal antibodies.     

Surface-enhanced Raman scattering immunoassays using a rotated capture substrate

A rapid, sensitive format for immunosorbent assays has been developed to meet the increasing levels of performance (i.e., reduction of incubation times and detection limits) demanded in the medical, veterinary, and bioterrorism prevention arenas. This paper introduces the concept of a rotating capture substrate as a facile means to increase the flux of antigen and label to the solid-phase surface and thereby reduce assay time. To this end, a sandwich-type assay is carried out that couples the specificity of antibody-antigen interactions with the high sensitivity of surface-enhanced Raman scattering detection. To investigate this strategy, polyclonal anti-rabbit IgG was immobilized on a gold capture substrate via a thiolate coupling agent. The capture substrate, capable of controlled rotation, was then immersed in a sample solution containing rabbit IgG, which served as a model analyte. After binding the target IgG, the substrates were immersed and rotated in an extrinsic Raman label (ERL) labeling solution, which is composed of gold nanoparticles (60 nm) coated with an aromatic moiety as the Raman scatterer and an antibody as the biospecific recognition element. The effect of substrate rotation on both the antigen binding and ERL labeling steps was investigated. Implementation of optimized rotation conditions resulted in the reduction of assay times from 24 h to 25 min and a 10-fold improvement in the limit of detection. Finally, the developed protocol was applied to the detection of rabbit IgG suspended in goat serum, which served to assess performance in a biological matrix.     

Electrochemical and quartz crystal microbalance detection of the cholera toxin employing horseradish peroxidase and GM1-functionalized liposomes.

An ultrasensitive method for the detection of the cholera toxin (CT) using electrochemical or microgravimetric quartz crystal microbalance transduction means is described. Horseradish peroxidase (HRP) and GM1-functionalized liposomes act as catalytic recognition labels for the amplified detection of the cholera toxin based on highly specific recognition of CT by the ganglioside GM1. The sensing interface consists of monoclonal antibody against the B subunit of CT that is linked to protein G, assembled as a monolayer on an Au electrode or an Au/ quartz crystal. The CT is detected by a "sandwich-type" assay on the electronic transducers, where the toxin is first bound to the anti-CT-Ab and then to the HRP-GM1-ganglioside-functionalized liposome. The enzyme-labeled liposome mediates the oxidation of 4-chloronaphthol (2) in the presence of H2O2 to form the insoluble product 3 on the electrode support or the Au/quartz crystal. The biocatalytic precipitation of 3 provides the amplification route for the detection of the CT. Formation of the insulating film of 3 on the electrode increases the interfacial electron-transfer resistance, Ret, or enhances the electrode resistance, R', parameters that are quantitatively derived by Faradaic impedance measurements and chronopotentiometric analyses, respectively. Similarly, the precipitate 3 formed on the Au/quartz crystal results in a mass increase on the transducer that is reflected by a decrease in the resonance frequency of the crystal. The methods allow the detection of the CT with an unprecedented sensitivity that corresponds to 1.0 x 10(-13) M.     

Development of zeptomole and attomolar detection sensitivity of biotin-peptide using a dot-blot gold nanoparticle immunoassay

An ultrasensitive, simple, and fast immunoassay for biotin-peptide detection using gold nanoparticles conjugated with antibodies has been developed. Biotin was covalently attached to a peptide and the biotin-peptide bound on a nitrocellulose membrane. Antibody-coated gold nanoparticles bound to the biotin-peptide formed red dots. With this method, 100 amol of the biotin-peptide was detected and no immunogold was bound to the membrane in the absence of biotin. The relative intensity of each dot was scored using Quantity One, a quantitative analysis software program. The linear working range of this assay was between 1 pmol and 1 micromol. The assay sensitivity was increased by silver enhancement to 100 zmol, and the linear working range was between 100 zmol and 100 fmol. This assay can be extended to detect target molecules, such as dioxin, digoxin, mercury, and so on, with matched antibodies and has potential broad applications in immunoassay.     

Bioconjugated fluorescent zeolite L nanocrystals as labels in protein microarrays

Zeolite L nanocrystals, as inorganic host material containing hydrophobic fluorophore N,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide in the unidirectional channels, are developed as new labels for biosensor systems. The external surface of the particles is modified with carboxylic acid groups for conjugation to primary amines of biomolecules such as antibodies. Anti-digoxigenin (anti-DIG) is selected to be immobilized on zeolite L via N-hydroxysulfosuccinimide ester linker. Together with DIG, it serves as a good universal binding pair for diverse analyte detection owing to the high binding affinity and low background noise. The conjugates are characterized by the dynamic light scattering technique for their hydrodynamic diameters and by enzyme-linked immunosorbent assay for antigen-antibody binding behavior. The characterizations prove that anti-DIG antibodies are successfully immobilized on zeolite L with their binding activities maintained. The microarray fluorescent sandwich immunoassay based on such nanocrystalline labels shows high sensitivity in a thyroid-stimulating hormone assay with the lower detection limit down to the femtomolar range. These new fluorescent labels possess great potential for in vitro diagnostics applications.