Correlation between Babesia Species Affecting Dogs in Taiwan and the Local Distribution of the Vector Ticks.

The objective of our study was to survey Babesia infection rates by PCR and tick species on stray dogs to correlate the distribution of Babesia with the distribution of ticks infesting dogs in Taiwan. Three hundred eighty-eight blood samples and 3037 ticks were collected from 388 roaming, and free-ranging owned dogs at residential sites in Taiwan between January 2015 and December 2017. The prevalence of B. gibsoni and B. vogeli was 15.7% (61/388) and 9.5% (37/388), respectively. Most positive B. gibsoni dogs were found in the northern part of the country 56/61 (91.8%), whereas a few were found in the middle 5/61 (8.2%). Babesia vogeli infection rates were 10%, 3.6%, and 18.2% in the northern, central, and southern regions, respectively. Five species of ticks were found: Rhipicephalus sanguineus (throughout Taiwan), Rhipicephalus haemaphysaloides (in the north), Haemaphysalis hystricis (in the north and middle of Taiwan), and Amblyomma testidunarium and Ixodes ovatus (both in the north). None of the dogs in the south were infected with B gibsoni, which correlated with the absence of H. hystricis, a tick recently identified as the local vector for B gibsoni. Babesia vogeli was more equally distributed, coinciding with R. sanguineus, a tick that is present throughout Taiwan. Anaemia was detected in 86.9% of infected dogs; among these dogs, approximately 19.7% showed severe anaemia (HCT < 20). These findings provide useful advice for owners regarding outdoor activities with their dogs and local veterinarians with a regional differential diagnosis of babesiosis in Taiwan.

Synthesis and properties of the kojic acid dimer and its potential for the treatment of Alzheimer's disease.

The kojic acid dimer (KAD) is a metabolite derived from developing cottonseed when contaminated with aflatoxin. The KAD has been shown to exhibit bright greenish-yellow fluorescence, but little else is known about its biological activity. In this study, using kojic acid as a raw material, we developed a four-step synthetic route that achieved the gram-scale preparation of the KAD in approximately 25% total yield. The structure of the KAD was verified by single-crystal X-ray diffraction. The KAD showed good safety in a variety of cells and had a good protective effect in SH-SY5Y cells. At concentrations lower than 50 µM, the KAD was superior to vitamin C in ABTS+ free radical scavenging assay; the KAD resisted the production of reactive oxygen species induced by H2O2 as confirmed by fluorescence microscopy observation and flow cytometry analysis. Notably, the KAD could enhance the superoxide dismutase activity, which might be the mechanism of its antioxidant activity. The KAD also moderately inhibited the deposition of amyloid-ß (Aß) and selectively chelated Cu2+, Zn2+, Fe2+, Fe3+, and Al3+, which are related to the progress of Alzheimer's disease. Based on its good effects in terms of oxidative stress, neuroprotection, inhibition of Aß deposition, and metal accumulation, the KAD shows potential for the multi-target treatment of Alzheimer's disease.

Clinical characteristics of naturally Babesia gibsoni infected dogs: A study of 60 dogs.

Babesia gibsoni is increasingly recognized globally as a cause of canine tick-borne anemic disease; however, only a few clinical reports of naturally acquired infection are available. In this systematic study of dogs presenting with B. gibsoni infection, clinical and laboratory data were collected for dogs with PCR-confirmed B. gibsoni infection admitted to the National Taiwan University Veterinary Teaching Hospital (NTUVH) from January 2014 through December 2015. Of the 60 dogs recruited, 20 (33.3%) had concurrent disease and 40 (66.7%) had only B. gibsoni infection. The severity of anemia in B. gibsoni infected dogs with concurrent or without concurrent infection was not significantly different. The most commonly observed hematological abnormalities were anemia (49/60, 81.7%) and thrombocytopenia (37/60, 61.7%). Of 49 dogs, 24 (49%) had severe to very severe anemia (PCV < 20%). The main biochemical abnormalities included hyperglobulinemia (28/53, 52.8%), hyperbilirubinemia (10/28, 35.7%) and elevated hepatic enzyme activity (7/48, 14.6%). In addition, 2 of the 60 the client-owned dogs and 5 of the 33 B. gibsoni-positive stray dogs were detected as having a naturally atovaquone-resistant strain, using the SimpleProbe® assay. The study results provide a useful clinical presentation of B. gibsoni infection and raise the issue of the naturally atovaquone-resistant strain currently existing in Taiwan.

Molecular evidence for the transovarial passage of Babesia gibsoni in Haemaphysalis hystricis (Acari: Ixodidae) ticks from Taiwan: a novel vector for canine babesiosis.

BACKGROUND: Babesia gibsoni is the predominant tick-borne protozoan blood parasite affecting dogs throughout the Oriental region. Babesia gibsoni is transmitted by Haemaphysalis longicornis, whereas a similar role has been suggested for Rhipicephalus sanguineus. Haemaphysalis longicornis does not occur in Taiwan, but R. sanguineus is widely distributed on dogs. However, clinical cases of babesiosis are mainly restricted to the northern part of the island. The discrepancy between tick distribution and clinical cases stimulated us to investigate the tick species distribution on dogs in northern Taiwan, with the aim to identify the local vector for canine babesiosis. METHODS: Ticks were collected from stray dogs or free ranging pet dogs in northern Taiwan between 2015 and 2017 and, after identification, were tested for the presence of tick-borne Babesia parasites using PCR and reverse line blot (RLB) hybridisation. Moreover, engorged ticks collected from the dogs were incubated at 28 °C to allow them to oviposit. Their subsequent larval progeny was also examined by PCR/RLB. RESULTS: A total of 1085 ticks collected from 144 stray dogs at different residential areas consisted of 5 different species: H. hystricis (n = 435), R. sanguineus (n = 582), R. haemaphysaloides (n = 43), Amblyomma testudinarium (n = 14) and Ixodes ovatus (n = 11) were identified. Babesia gibsoni DNA was detected in H. hystricis females (10.3%), males (7.0%) and in 2.6% of the nymphs. One R. sanguineus female and one A. testudinarium female tick also carried B. gibsoni DNA. DNA of B. gibsoni was demonstrated in 11 out of 68 (16.2%) batches of larval ticks derived from engorged H. hystricus ticks only. Babesia vogeli DNA was detected only in R. sanguineus females (2.6%) and males (2.4%). DNA of B. vogeli was detected in 13 out of 95 (13.7%) batches of larval ticks derived from engorged R.sanguineus females. CONCLUSIONS: Babesia gibsoni DNA was detected in the larval progeny of H. hystricis ticks only, whereas B. vogeli was restricted to the larvae of R. sanguineus. This provides evidence for transovarial passage of B. gibsoni in H. hystricis and evidence that this tick does act as the local vector for this parasite on dogs in northern Taiwan where most cases of babesiosis are reported. The vectorial capacity of R. sanguineus for babesiosis is probably restricted to the transmission of B. vogeli only.

A SimpleProbe(®) real-time PCR assay for differentiating the cytochrome b M121I mutation in clinical specimens from dogs infected with Babesia gibsoni.

Babesia gibsoni (B. gibsoni) causes a canine tick-borne disease worldwide. The substitution of methionine with isoleucine (M121I) in the cytochrome b (CYTb) gene of B. gibsoni was identified as being associated with atovaquone resistance. Rapid identification of the drug-resistant strain is required to select a more effective combination of drugs, e.g., from atovaquone and azithromycin (AA) to clindamycin, diminazene, and imidocarb (CDI) combination. A SimpleProbe(®) real-time PCR assay was designed to detect the single nucleotide polymorphism at nucleotide 363 in CYTb gene of B. gibsoni and the sensitivity and specificity were evaluated by comparing the results from the conventional DNA sequencing method. Eighty-nine clinical blood samples were collected and analyzed in parallel with the SimpleProbe(®) assay and DNA sequencing. The assay identified 50 of 54 nt363G samples and had a sensitivity of 92.6% and a specificity of 100%. Thirty nt363T samples were correctly identified, as well, with a sensitivity of 100% and a specificity of 73.2%. However, this assay identified only one of 17 nt363A samples; the other 16 samples were misidentified as nt363T. The sensitivity of the nt363A identification was only 5.9%, and the specificity was 100%. When detecting the M121I mutation, 42 of 42 mutant samples were identified, with a sensitivity of 100%, and 45 of 47 wild type samples were identified, with a specificity of 95.7%. In conclusion, the SimpleProbe(®) assay could be used to detect the M121I mutation of the B. gibsoni CYTb from clinical specimens. This assay provides a reliable and sensitive tool for differentiating between the atovaquone-resistant strain and the non-resistant strain.

Influenza A(H6N1) Virus in Dogs, Taiwan.

We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species.

An outbreak of feline infectious peritonitis in a Taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type II feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) infection. FCoV can be divided into serotypes I and II. The virus that causes FIP (FIPV) is believed to occur sporadically and spread infrequently from cat to cat. Recently, an FIP outbreak from an animal shelter was confirmed in Taiwan. FCoV from all the cats in this shelter were analyzed to determine the epidemiology of this outbreak. Thirteen of 46 (28.2%) cats with typical signs of FIP were identified. Among them, seven cats were confirmed by necropsy and/or histopathological examinations. Despite the fact that more than one FCoV was identified in this multi-cat environment, the eight FIP cats were invariably found to be infected with a type II FCoV. Sequence analysis revealed that the type II FIPV detected from fecal samples, body effusions and granulomatous tissue homogenates from the cats that succumbed to FIP all harbored an identical recombination site in their S gene. Two of the cats that succumbed to FIP were found to harbor an identical nonsense mutation in the 3c gene. Fecal shedding of this type II virus in the effusive form of FIP can be detected up to six days before death. Taken together, our data demonstrate that horizontal transmission of FIPV is possible and that FIP cats can pose a potential risk to other cats living in the same environment.

Full genome analysis of a novel type II feline coronavirus NTU156.

Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated with fatal feline infectious peritonitis (FIP). Despite nearly six decades having passed since its first emergence, different studies have shown that type II FCoV represents only a small portion of the total FCoV seropositivity in cats; hence, there is very limited knowledge of the evolution of type II FCoV. To elucidate the correlation between viral emergence and FIP, a local isolate (NTU156) that was derived from a FIP cat was analyzed along with other worldwide strains. Containing an in-frame deletion of 442 nucleotides in open reading frame 3c, the complete genome size of NTU156 (28,897 nucleotides) appears to be the smallest among the known type II feline coronaviruses. Bootscan analysis revealed that NTU156 evolved from two crossover events between type I FCoV and canine coronavirus, with recombination sites located in the RNA-dependent RNA polymerase and M genes. With an exchange of nearly one-third of the genome with other members of alphacoronaviruses, the new emerging virus could gain new antigenicity, posing a threat to cats that either have been infected with a type I virus before or never have been infected with FCoV.

The therapeutic efficacy of two antibabesial strategies against Babesia gibsoni.

Various combination strategies for treating Babesia gibsoni have been described. However, relapses after administering some combinations of antibabesial drugs and the presence of drug-resistant B. gibsoni still pose significant challenges to veterinarians. To compare the efficacy of a combination of clindamycin, diminazene, and imidocarb (CDI) to that of a combination of atovaquone and azithromycin (AA) for the treatment of B. gibsoni and to correlate drug efficacy with B. gibsoni mutations, 30 client-owned dogs with natural B. gibsoni infections were collected in the study. 17 dogs were treated with AA, and 13 dogs were treated with CDI combination. Hematological parameters were recorded on the day that the dogs were presented for treatment and during treatment. To detect the parasitic DNA, the B. gibsoni 18S rRNA gene was amplified, and to analyze the mutations, the cytochrome b (CYTb) gene was sequenced. The therapy duration for all of the dogs that recovered was 23.3±7.8 days in the AA group and 41.7±12.4 days in the CDI group. Nine of the 17 dogs in the AA group and 11 of the 13 dogs in the CDI group completely recovered. Seven dogs in the AA group and 2 dogs in the CDI group relapsed after treatment. The M121I mutation in the B. gibsoni CYTb gene was detected in all of the samples that were collected from AA-relapsed and AA-nonremission dogs. The dogs in the CDI group exhibited higher recovery rates and lower relapse rates during treatment for B. gibsoni infection. In addition, the detected M121I mutation was associated with AA treatment. The CDI combination is a promising alternative treatment strategy for B. gibsoni.

Multiarmed star-like platinum nanowires with multienzyme assembly for direct electronic determination of carcinoembryoninc antigen in serum.

A new electrochemical immunoassay strategy for direct detection of carcinoembryoninc antigen (CEA) in serum was developed by using multiarmed star-like platinum nanowires (PtNWs) with biomolecular assembly as signal tags on an anti-CEA-functionalized graphene sensing platform. Initially, the PtNWs were synthesized via a wet chemical method, and then the synthesized PtNWs were used for the co-immobilization of CEA and horseradish peroxidase (HRP). Compared with platinum nanoparticles, the prepared PtNWs could provide a large room for the conjugation of HRP and CEA. With a competitive-type immunoassay format, the assay was performed in two types of supporting electrolytes including new born cattle serum (NBCS) and acetate buffer solution (ABS, pH 5.5), respectively. Similar detection limit (LOD) of 5.0 pg mL(-1) vs. 1.0 pg mL(-1) but narrower dynamic working linear range of 0.01-60 ng mL(-1) vs. 0.002-80 ng mL(-1) was obtained toward CEA standards in the NBCS compared to the ABS. The intra-assay coefficients of variation (CVs) were 4.3%, 8.6%, and 6.2% at 0.05, 10, and 40 ng mL(-1) CEA, respectively, while the inter-assay CVs were 7.6%, 10.5%, and 8.9% at the above-mentioned levels, respectively. In addition, the selectivity and stability of the electrochemical immunosensor were acceptable. Importantly, the developed method was used to assay clinical serum specimens, receiving a good relation with those obtained from the referenced method.

Ultrasensitive aptamer-based multiplexed electrochemical detection by coupling distinguishable signal tags with catalytic recycling of DNase I.

This work reports an aptamer-based, disposable, and multiplexed sensing platform for simultaneous electrochemical determination of small molecules, employing adenosine triphosphate (ATP) and cocaine as the model target analytes. The multiplexed sensing strategy is based on target-induced release of distinguishable redox tag-conjugated aptamers from a magnetic graphene platform. The electronic signal of the aptasensors could be further amplified by coupling DNase I with catalytic recycling of self-produced reactants. The assay was based on the change in the current at the various peak potentials in the presence of the corresponding signal tags. Experimental results revealed that the multiplexed electrochemical aptasensor enabled the simultaneous monitoring of ATP and cocaine in a single run with wide working ranges and low detection limits (LODs: 0.1 pM for ATP and 1.5 pM for cocaine). This concept offers promise for rapid, simple, and cost-effective analysis of biological samples.

Specific immunoreaction-induced controlled release strategy for sensitive impedance immunoassay of a cancer marker.

A simple and facile impedance immunoassay strategy for sensitive detection of alpha-fetoprotein (AFP), as a model cancer marker, was developed by using target-induced release of nanogold particle-labelled anti-AFP antibodies from polyvinylpyrrolidone-coated magnetic carbon nanotubes.

Magneto-controlled electrochemical immunosensor for direct detection of squamous cell carcinoma antigen by using serum as supporting electrolyte.

A new magneto-controlled microfluidic device for direct electrochemical determination of squamous cell carcinoma antigen (SCC-Ag) in serum was designed by using anti-SCC antibody (SCC-Ab)-functionalized magnetic mesoporous nanogold/thionine/NiCo(2)O(4) hybrid nanostructures as immunosensing probes (P(1)-Ab) and horseradish peroxidase-SCC-Ab conjugates-labeled nanogold/graphene nanosheets as signal tags (P(2)-Ab). In the presence of the analyte SCC-Ag, the sandwich immunocomplex was formed between the immunosensing probes and the signal tags. With the aid of an external magnet, the formed immunocomplex was attached to the microfluidic device. The assay was implemented in newborn calf serum (NBCS) containing 2.5 mM H(2)O(2) based on the labeled peroxidase on the P(2)-Ab toward the catalytic reduction of H(2)O(2). Under optimal conditions, the increase in the current was proportional to the concentration of SCC-Ag from 2.5 pg/mL to 15 ng/mL. The detection limit (LOD) was 1.0 pg/mL SCC-Ag at 3s(B). The electrochemical immunoassay displayed an acceptable precision, selectivity and stability. Clinical serum specimens were assayed with the method, and the results were in acceptable agreement with those obtained from the referenced electrochemiluminescent method. Importantly, the method can be suitable for on-line use in the mass production of miniaturized lab-on-a-chip devices and open a new opportunity for protein diagnostics and biosecurity.

Electrochemical detection of hepatitis C virus with signal amplification using BamHI endonuclease and horseradish peroxidase-encapsulated nanogold hollow spheres.

A novel electrochemical method to detect hepatitis C virus was developed based on site-specific cleavage of BamHI endonuclease and enzymatic signal amplification with horseradish peroxidase-encapsulated nanogold hollow spheres.

Sensitive electrochemical immunoassay of carcinoembryonic antigen with signal dual-amplification using glucose oxidase and an artificial catalase.

A new dual-amplification strategy of electrochemical signal based on the catalytic recycling of the product was developed for the antigen-antibody interaction by glucose oxidase (GOD)- conjugated gold-silver hollow microspheres (AuAgHSs) coupled with an artificial catalase, Prussian blue nanoparticles (PB), on a graphene-based immunosensing platform. The first signal amplification introduced in this study was based on the labeled GOD on the AuAgHSs toward the catalytic oxidation of glucose. The generated H(2)O(2) was catalytically reduced by the immobilized PB on the graphene nanosheets with the second amplification. With a sandwich-type immunoassay format, carcinoembryonic antigen (CEA) was monitored as a model analyte by using the synthesized AuAgHSs as labels in pH 6.0 phosphate buffer containing 10mM glucose. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005-50 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) CEA (at 3σ). Both the intra- and inter-assay coefficients of variation (CVs) were lower than 10%. The specificity and stability of the immunosensor were acceptable. In addition, the assay was evaluated for clinical serum specimens, and received a good correlation with those obtained by the referenced electrochemiluminescent (ECL).

An organic-inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels.

A new electrochemical immunoassay of alpha-fetoprotein (AFP) was developed on an organic-inorganic hybrid nanostructure-functionalized carbon electrode by coupling with magnetic bionanolabels. Multi-walled carbon nanotubes (CNTs), single-stranded DNA, thionine and AFP were utilized for the construction of the immunosensor, while the core-shell Fe(3)O(4)-silver nanocomposites were employed for the label of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-AgFe). Electrochemical measurement toward AFP was carried out by using magnetic bionanolabels as traces and H(2)O(2) as enzyme substrate with a competitive-type immunoassay mode. Experimental results indicated that the immunosensors with carbon nanotubes and DNA exhibited better electrochemical responses than those of without carbon nanotubes or DNA. Under optimal conditions, the electrochemical immunosensor by using HRP-anti-AFP-AgFe as signal antibodies exhibited a linear range of 0.001-200 ng mL(-1) AFP with a low detection limit of 0.5 pg mL(-1) at 3s(B). Both intra- and inter-assay coefficients of variation were 7.3%, 9.4%, 8.7% and 10.2%, 7.8%, 9.4% toward 0.01, 30, 120 ng mL(-1) AFP, respectively. The specificity and stability of the electrochemical immunoassay were acceptable. In addition, the methodology was validated for 12 clinical serum specimens including 9 positive specimens and 3 normal specimens, receiving a good correlation with the results obtained from the referenced electrochemiluminescence assay.

Gold-silver-graphene hybrid nanosheets-based sensors for sensitive amperometric immunoassay of alpha-fetoprotein using nanogold-enclosed titania nanoparticles as labels.

A new sandwich-type electrochemical immunosensor with enhanced sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) in biological fluids by using nanogold-enclosed titania nanoparticle (AuTi)-labeled secondary antibody on a gold-silver-graphene hybrid nanosheet (AuAgGP)-functionalized glassy carbon electrode (GCE). The presence of the AuAgGP nanosheets not only enhanced the immobilized amount of biomolecules, but also improved the electrochemical properties of the immunosensor. With the aid of AuTi nanolabels, the electrochemical signal was greatly amplified in comparison with pure nanogold or titania-based labels. Under optimal conditions, the sensitivity and dynamic range of the immunosensor were evaluated by using the labeled horseradish peroxidase on the AuTi as trace and H(2)O(2) as enzyme substrate, and exhibited a wide dynamic range of 0.001-200 ng mL(-1) with a low detection limit (LOD) of 0.5 pg mL(-1) AFP (at 3σ). Both the intra- and inter-assay coefficients of variation were less than 10%. The current of the immunosensor at 13th day was as much as 90% of the initial current. In addition, the methodology was evaluated for 8 positive serum specimens obtained from hepatocarcinoma patients and 19 negative sera, and validated with the commercially available Roche 2010 Electrochemiluminescent (ECL) Automatic Analyzer. No significant differences at the 95% confidence level were encountered between two methods.

Carbon nanotube-based symbiotic coaxial nanocables with nanosilica and nanogold particles as labels for electrochemical immunoassay of carcinoembryonic antigen in biological fluids.

A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol-gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO(2)/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO(2)/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO(2)/CNTs as detection antibodies. To embody the advantages of the protocol, the analytical properties of variously modified electrodes were compared in detail on the basis of different nanolabels. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12 ng mL(-1) in pH 5.5 HAc-NaAc containing 5mM H(2)O(2). At a signal-to-noise ratio of 3, the detection limit (LOD) is 5 pg mL(-1) CEA. Intra- and inter-assay coefficients of variation were below 9.5%. Meanwhile, the selectivity and stability of the immunosensor were acceptable. In addition, the technique was evaluated by spiking CEA standards in pH 7.4 PBS and with 35 clinical serum specimens, receiving excellent accordance with results from commercially available electrochemiluminescent enzyme-linked immunoassay.

A graphene-based Au(111) platform for electrochemical biosensing based catalytic recycling of products on gold nanoflowers.

A novel, enzyme-free amperometric immunoassay of biomarkers with sensitive enhancement was designed by using gold nanoflower-labeled detection antibodies toward the catalytic reduction of p-nitrophenol and redox cycling of p-aminophenol on a graphene-based Au(111) platform.

Enzyme-free electrochemical immunoassay with catalytic reduction of p-nitrophenol and recycling of p-aminophenol using gold nanoparticles-coated carbon nanotubes as nanocatalysts.

A novel enzyme-free sandwich electrochemical immunoassay with an ultrahigh sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) using carbon nanotube-enriched gold nanoparticles (CNT-AuNPs) as nanolabels/nanocatalysts on anti-AFP/glutaraldehyde/thionine-modified glassy carbon electrodes (GCEs). The assays were carried out in a pH 8.0 acetic acid-buffered solution containing 6 mM p-nitrophenol (NP) and 6mM NaBH(4) after the formation of the sandwich-type immunocomplex. Initially, the NP molecules were reduced to p-aminophenol (AP) by the catalysis of the immobilized gold-nanoparticle labels on the CNT-AuNPs with the aid of NaBH(4), then the generated AP molecules were electrochemically oxidized to p-quinone imine (QI) by an electron mediator of thionine, and then the oxidized QI molecules were reduced back to APs by NaBH(4). The redox cycling of AP and QI continuously increased the signaling, leading to a high sensitivity. Compared with individual gold-nanoparticle labels, the immunosensor using CNT-AuNPs as labels displayed a wider linear range of 8.0×10(-7)-2.0×10(2) ng/mL with a lower detection limit (LOD) of 0.8 fg/mL AFP at a signal-to-noise ratio of 3, which was lower 6 orders than that of commercially available ELISA. Intra-and inter-assay coefficients of variation were below 10%. In addition, the assay was evaluated with clinical serum samples, and no significant differences at the 5% confidence level were encountered in the analysis of real samples between the proposed immunoassay and commercially available Roche 2010 Electrochemiluminescent Automatic Analyzer for determination of AFP.