A novel sensitive immunoassay by nucleic acid barcode dot and its application in the detection of prostate-specific antigen.
BACKGROUND
The sensitivity and selectivity of traditional methods limits ultramicro detection of proteins. Bio-barcode amplification detection methods based on nanotechnology enables ultramicro detection of protein. However, bio-barcode amplification detection depends on the oligonucleotides being fixed on a glass chip. It also requires specialized equipment, which limits its application. We introduce a nano-nucleic acid barcode dot detection technology to determine ultramicro concentrations of protein. The method is simple, quick and accurate.


METHODS
Magnetic probe (IgG-M) and dual-labeled gold nanoparticle bio-probe (IgG-Au-DNA) were prepared. Protein was captured using a sandwich assay technique and magnetic separation was used. The DNA barcode was released with dithiothreitol (DTT) and detected directly without the requirement for polymerase chain reaction (PCR). Serum prostate-specific antigen (PSA) from 135 patients was detected with this method and compared with enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).


RESULTS
Each IgG-Au-DNA could be covered with 138+/-47 oligonucleotides and 11+/-3 antibodies. The IgG-M could bind 118 mug of antibody per mg. The sensitivity of nano-nucleic acid barcode dot detection technology might allow detection of 1 fg/mL. There were no significant differences in serum PSA from 135 patients when comparing the three methods (compared with ELISA, r=0.950; and with RIA, r=0.967).


CONCLUSIONS
The nucleic acid barcode dot method does not require special equipment or complex procedures, but its detection limit is 2-3 orders of magnitude lower than ELISA.
Kong
Xiao-Li
XL
Clinical Medical Research Center, The 2nd Clinic Medicine College (Shenzhen People's Hospital), Jinan University, Shenzhen, PR China.
Qi
Hui
H
Zhou
Han-Xin
HX
Ren
Li-Li
LL
Deng
Chong-Yan
CY
Li
Fu-Rong
FR
D000328:Adult; D000368:Aged; D015342:DNA Probes / Q000032:analysis*; D004229:Dithiothreitol / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006046:Gold / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007075:Immunoglobulin M / Q000737:chemistry; D057230:Limit of Detection; D008280:Magnetics; D008297:Male; D053768:Metal Nanoparticles / Q000737:chemistry; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000235:genetics; D045663:Quantum Dots; D011863:Radioimmunoassay
D015342:DNA Probes; D007075:Immunoglobulin M; D006046:Gold; D017430:Prostate-Specific Antigen; D004229:Dithiothreitol