Localized Surface Plasmon Resonance Biosensor for Detection of Serum Prostate Specific Antigen in Prostate Cancer Patients

1Department of Immunology, Shahid Sadoughi University of Medical Sciences & Health Services, Yazd, Iran. 2Faculty of New Science and Technology, University of Tehran, Iran. 3Medical Nano-Technology & Tissue Engineering Research Center, Department of Tissue Engineering and Regenerative Medicine, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Prostate cancer is a major health issue in the world .It is third most common cancer , occurring mostly in men older than 50 years 1 .earlydetection of the prostate cancer is important for decreasing the mortality rates.Human Prostatespecific antigen (PSA) was detected in 1980 2 .PSA a 32 -33 kd single -chain glycoprotein is found in serum, seminal plasma, benign hyperplastic and prostatic fluids, so it is a well-known tumor biomarker for prostate cancer screening and monitoring after treatment.the serum of men have naturally trace levels of PSA but prostate cancer tumor growth leads to increasing levels of PSA in serum 3,4 ., A PSA level below 4ng/ml is regarded as negative ;between 4-10 ng/ml is gray zone and A total PSA level (i.e.free PSA plus ACT-bound PSA) above of 10 ng/ml or higher is regarded as positive and a highly probable indication for prostate cancer 5 .
At the present, most tumor biomarker is analyzing with large and automated instrument and most of these analyzers are time-consuming and high cost 7 .The using of biosensor as diagnostics tools is one of the way to improve of some of problems such as sensitivity , accuracy ,fast ,easy to use and inexpensive methods of analysis biosensor can play a great role for in the early detection cancer 7 .Biosensor is diagnostics device for analyzing of biomaterial sample by converting of biological response into an electrical signal .thereare many various biosensor that include calorimetric, potentiometric, aerometric, piezoelectric and optical biosensors [8].Localized surface Plasmon resonance (LSPR) technique based on gold nano-particles is good methods because it has good sensitivity, good selectivity and short assay time 9 .
LSPR is a noble characteristic of metallic nanoparticle, caused by the collective oscillation of free electrons by incidence light.Nanoplasmonic phenomenon is an intensive absorption and scattering of light come from in 3-dimensional metallic nanoparticle 6,10 .Shape, orienta-tion, size, composition and local dielectric environment of nanoparticles are factors which can effect of LSPR extinction spectrum in in the ultraviolet (UV)visible region 9 .
bio-molecular interactions can change of the local refractive index near the nanoparticle sur-face and the peak wavelength of the LSPR extinction spectrum (ëmax) is very sensitive environmental refractive indexes, so This opti-cal property enables metallic nanoparticles to serve in different fields such as medical diagnosis, environmen-tal monitoring, drug screening and food safety [11][12][13][14] .
Moreover, the LSPR biosensor has advantages in biomarker detection, rapid test time, low cost, and direct assay format, unlike approaches; and compared with the traditional immunoassay(e.g., ELISA, CLA) 15 .

Patients and samples
The human serum samples from prostate cancer patients were collected from shahid beheshti University Hospital (Tehran, People's Republic of Iran).Written informed consent was not obtained.Sera were isolated from whole blood samples via centrifugation at 3,000 rpm for 15 minutes, and subsequently kept frozen at -80°C until analysis.

Instrumentations
The optical absorption spectra were recorded with a UV-3600 spectrophotometer (England).The TEM images were measured with a JEM-200CX transmission electron microscope (Philips, Japan).The samples for taking TEM imagines were prepared by evaporation of a drop of nanoparticles/bio conjugates solution on carbon films supported on standard copper grids.The exact pH value was obtained using a PP-50 pH meter (Sartorius, Germany)

Preparation of Gold Nanoparticles
In a typical synthesis route, GNPs were synthesized with diameters in the range of 12-22nm through sodium citrate reduction of hydrogen tetrachloroaurate (III) (HAuCl43H2O).Briefly, 50mL of 0.3mM (HAuCl43H2O) was brought to a vigorous boil with stirring; subsequently, 5mL of 5mM sodium citrate was added rapidly to the solution.This solution was boiled for another 15 min, during which time the solution changed color from pale yellow to deep red.The solution left to cool to room temperature, and then stored in a refrigerator until to use.The resulting colloidal gold solutions were characterized by visible absorption spectra and transmission electron microscope.
11-MUA assembles on citrate stabilized gold nanoparticles according to the procedure described in Ref. [41]In brief, 9.0 mL phosphate buffer (10 mM ,pH 7.0with 0.2 mg/mL Tween-20) was added to 18.0 mL gold nanoparticles and the obtained solution was allowed to stand for at least 60 min.Then 18 mL 11-MUA solution (0.5 mM in 1:3 alcohol/H2O) was added the mixture, followed by a gentle shake for 5 h.after shaking centrifugation was carried out in 1.5 mL Tubes using a 5415D centrifuge (Eppendorf, Germany) at a speed of 16000 × g rcf.Every tube contained 1.0 mL mixture.The centrifugation procedure was kept for 30 min and repeated three times.The supernatant containing alkane thiol of every tube was removed, while the sediment was resuspended in 1 mL phosphate buffer (10 mM, pH 7, with 0.2 mg/mL Tween-20).
The activation of hydroxyl-terminal groups was performed with the presence of EDC and NHS [16-18].In detail, a mixture solution (200µL) of freshly prepared 200 mM EDC and 50 mM NHS was added to 11-MUA modified gold nanoparticles solution and the mixture was allowed to incubate for 10 min.The resulted mixture in every tube was centrifuged to remove unreacted EDC/ NHS and dispersed in 1 mL phosphate buffer as described above.This mixture were finally dispersed under ultra-sonication.

Attachment with PSA Antibody
Mouse anti-PSA antibody was coupled to the gold nanoparticles via displacing the NHS group with the lysine residues of the antibody.36As such, 200 µL solution of mouse anti-PSA antibody (4 µg/mL) was added to the suspension of gold nanoparticles with NHS ester groups in every tube.The mixture was then incubated at 4 °C for at least 3 h.After centrifugation at 16,000×g for 20 min and removal of the supernatant, the sediment in every tube was dispersed in 1 mL of 10 mM phosphate buffer (pH 7.4).

Serum sample detection
Under the room temperature, every tube contained 0.5 mL Antibody Coated Gold Nanoparticles was added 50 µL serum sample and incubate for 20 min and LSPR peak serum sample was measured by UV-visible spectrophotometer ranging from 400 nm to 800 nm.

RESULTS AND DISCUSSION
To figure out of optical properties of Nano-particles and Particle size distribution and geometry and Surface charge of nanoparticles, optical absorption spectroscopy, TEM and DLS were performed.Figure .1 shows the optical absorption spectra of different AuNPs in each stages, in which the curve peak shows the characteristic surface Plasmon resonance (SPR) of AuNPs 19 .
The SPR of Pure gold nanoparticles with red color (curve A) is about 520 nm.The and the SPR shifts to 523 nm after chemisorption of 11MUA and color of gold nanoparticles changes to dark red (curve B).by Au-S It is obvious that a formed dielectric monolayer of 11-MUA had been on around AuNPs and alkane thiol molecules are adsorbed on surface of gold nanoparticles by interaction between Au-s 20,21 .theCOOH terminal of 11-MUA , the SPR peak at 543 nm (curve c) .The obvious red shifting and changing color suggest that the NHS-terminated gold nanoparticles with a partial aggregation colloidal particle aggregation because the NHS esterification of terminal carboxyl group can charge neutralization and lose ionizable carboxyl groups on nanoparticle surface during the activation using of EDC/NHS 22,23 .finally, the gold nanoparticles/antibody solution (curve d) displays a SPR peak at 550nm.The SPR peak shifting and purple color final bioconjugate solution is only 27 nm compared with MUAmodified AuNPs, suggest that gold nanoparticles with antibody had coupled.
Figure .2shows the FT-IR spectra of AuNPs.For citrate-stabilized AuNPs, absorbance at about 1690 cm -1 represents the citrate component by an evidential carboxylate groups (curve a) [23].Peaks obtained from FTIR analysis represents a good functional groups on the surface of gold nanoparticles.For MUA modiûed AuNPs, the distinct peaks appeared at 2849 cm -1 are ascribed to the vibrational stretches of -CH2 groups of long alkane chains, For NHS-terminated AuNPs, the peak at 1742 cm -1 assigned to the succinimidyl carbonyl group, and the two weak peaks at 1830 and 1782 cm -1 assigned to the band splitting of the ester carbonyl C=O stretching vibration (curve b) 23 .For the Antibody/AuNPs bioconjugates, the peaks at 1650 and 1540 cm -1 assigned to the secondary  25 .In this study, the aggregation ratios was obtained 1.38 which indicated stability of nanoparticles.
The LSPR sensor were incubated in different concentrations of standard total PSA solution ranging from 1 ng/ml to 50 ng/ml under optimal conditions, and LSPR peak were obtained with peak shifts recorded.Mean value and standard deviation of "ëmax were calculated, since each concentration measurement was repeated three times.As shown in Figure 5, the LSPR "ëmax values increased stepwise with increasing total PSA concentrations and were Exponential equation.Correlation coefficient (R) of 0.9987 and the limit of detection (LOD) in the present method is 0.9 ng/ mL which was obtained by the equation LOD=3 (SD/S).The average sensitivity of the LSPR method for total PSA 1.1 ng/ml was obtained.
In this study, we investigated the practicability of the proposed LSPR sensor for detecting Total PSA in human serum.Total PSA concentration of each serum sample was calculated according to the Exponential regression equation.A series of six serum samples from prostate cancer patients were analyzed and the results were compared with chemiluminescence assay (Fig. 6).
The selectivity is known as a key parameter in LSPR sensors.To investigate the selectivity of this sensor, the influences of various tumor marker (CEA, CA125, CA19-9, BHCG, and Alb) in the certain concentration were tested (Fig. 7).The results indicate that, no significant shift is observed in samples containing other tumor marker.
The results demonstrate that in the present method a high selectivity for the sensing of total PSA [25][26][27][28][29] .
To study the effect of the time stability, the detection of total PSA at different times are performed (Fig. 8).The results show that after 20 day (at 4°C) the sensor response change is only about 32% 31 .

CONCLUSION
This method can be successfully used as an LSPR-based sensor for total PSA.We fabricated antibody covalently gold nanoparticles using 11-MUA as the linker.The localized surface plasmon resonance (LSPR) as a tool for interaction between antigens and antibodies immobilized on the surface of gold nanoparticles were used .The present results show that when the nanoparticles such as AuNps are used, the sensitivity and selectivity of LSPR sensor for sensing of total PSA can be improved.In addition, the sensor also shows a good stability.The amount of average sensitivity and low detection limit of the LSPR sensor for total PSA respectively 1.1 and 0.9 ng / ml was calculated .This method is rapid, selective, and highly sensitive assay and it has potential applications in assay for other tumor marker.