Gold nanoparticles enhanced fluorescence for highly sensitive biosensors based on localized surface plasmon resonance applied in the determination C-reactive protein

Introduction: C-reactive protein (CPR) is known as an inflammation marker related to numerous pathology. Optical biosensor based on the fluorescence dyed is widely used in diagnosis. There are still limitations on fluorescence signal detection due to the photobleaching effect. The localized surface plasmon resonance (LSPR) performed by gold nanoparticles (Au NPs) is testified to enhance photo-signal gathered from the dye molecules. The fluorescence enhancement by the effectofAuNPscoatingontheluminescenceisdestinedastheevolutionoflabel-basedbiosensing. Methods: In this study, Au NPs were used for their significant optical properties and biocompati-bility additionally. The seed-mediated synthesis method provided stable NPs with all the essential qualities. A series of modification steps were done on a glass substrate before the bio-bonding for fluorescence-based sensing by a transmission mode (T-mode) detection system introduced in Viet Nam. Results: The synthetic Au NPs in the nanosphere structure evinced the absorbance at a maximum wavelength is 521 nm. All the followed alterations showed the accomplishment in forming the in need linking proved through the basic analysis methods. Finally, CRP with the Alexa 488 dye was observed for average at 4.8 folds of enhancement factor compared between the Au NPs coating and non-coating substrate detected by the T-mode system. The low coefficient of variation at under 0.7% appeared for the repeatability and stability of this sensor. Conclusion: The T-mode system's completely modern approach combined with the LSPR applied in fluorescence sensors enhanced is developed successfully. The future prospect of this designed sensing method is promising by changing the materials' structures and ingredients. issues determine such as lymphoma, intestinal hem-orrhage, or rheumatoid arthritis and access to their treatment respond 12 . Further CRP quantitative studies were applied various methods, including electro-chemical immunoassay (ECL), photothermal system (PTB), vertical flow immunoassay (VFA), and so on gained some remarkable results 13 . This work aims to demonstrate the extraordinary im-provement of luminescence signal exploits brought by gold nanoparticles aid for a potential optical biosensor. The Alexa 488 fluorophore molecules stained on CRP are eager to strengthen and stable under the as-sistance of nano metallic coating.


INTRODUCTION
Label-based biosensors are widely applied in life science and beyond. One of the most powerful utilizations of that rapid and high-performance method is in biomedical fields such as diagnosis of infectious diseases, cancer detection, DNA determination 1-3 , etc. To increase the sensitivity for lower the test limit of detection (LOD), the potential metal nanoparticles (NPs) were used since their outstanding behavior in optical, electrochemical, plasmonic, and radiative properties [4][5][6] . In this paper, gold nanoparticle (Au NPs) synthesized by Turkevich method 7 was investigated for its localized surface plasmon resonance (LSPR) effects in other signal enhancement for a labeled based optical biosensor system applied in Creactive protein (CRP) detection.
Besides other featured optical properties that were mentioned even from the Lycurgus Cup's invention back to the 4 th century A.D, localized surface plasmon resonance (LSPR) is an attractive optical phenomenon most found in the noble metal nanostructure. The exceptional ability proved in the absorption of the special incoming light then converts the photon energy into the electrons' oscillation before scattering it with another collective-wavelength outcome light 8 . Therefore, the optical occurrence consisting of photobleaching does not occur as if LSPR happened 9 . Based on the difference in the nanoparticles size, shape, ingredient, and interspace, a large adjustment of LSPR was provided mostly in the maximum absorbance wavelength under the excitation light 10 . Gold was used in our study since its chemical stability forces a wide range of applications and is currently being attended in biosensors 11 . CRP is known as an inflammation marker significant aggregation in the liver and recommended testing in postoperative infection monitor cases, pathological issues determine such as lymphoma, intestinal hemorrhage, or rheumatoid arthritis and access to their treatment respond 12 . Further CRP quantitative studies were applied various methods, including electrochemical immunoassay (ECL), photothermal system (PTB), vertical flow immunoassay (VFA), and so on gained some remarkable results 13 . This work aims to demonstrate the extraordinary improvement of luminescence signal exploits brought by gold nanoparticles aid for a potential optical biosensor. The Alexa 488 fluorophore molecules stained on CRP are eager to strengthen and stable under the assistance of nano metallic coating.

PREPARATION OF GOLD NANOPARTICLES (AU NPS)
The synthesis of Au NPs was completed by the seedmediated method with NaCtr roled as the reductant for its rapid and simple protocol 14 . A colloidal solution of gold nano seed was quickly assembled at high temperatures by the ingredient reaction. The next stage of growing seed was continuous right after the cooling down period while the light was avoided to prevent photoreduction. The appropriate volume of grown solution was synthesized from the remaining scattering in the ultrasonic bath and finally stored in the required condition.

PREPARATION OF THE MODIFIED SUBSTRATES
The clean glass substrates underwent a pre-set process of the CUTE plasma cleaner from Femto Science, Korea, for the silanol forming before being treated by 3% APTES in ethanol to fabricate amine-rich surfaces. Right after, two separate antibody bonding methods were performed. Figure 1a illustrates a five-step no nano metal coating process with the direct antibody linking to the carboxyl from SA treatment. On the other hand, Figure 1 b shows the advanced Au NPs coating process by simply immersion in the nanoparticle storing condition. 11-MUA was used for carboxyl modification after the nanometal middle layer and responded to the CRP@Alexa 488 coupling.

Fabrication of PDMS microfluidic LSPR device
A PDMS round-shaped mask was placed on the glass substrate after fabrication from mixing the base and the curing agent then heating up to solid 15 . The mask played a role in fitting the light pathway from the optical detection system and limiting chemical loss in the modification.

Analytical Methods
The optical property of the nanoparticles was analyzed by the V-730 Ultraviolet-visible spectroscopy (UV-Vis) from JASCO, in the range from 400 nm to 800 nm for determination of the LSPR accordant maximum absorbance wavelength. The accomplishment of the surface modification was assessed by Fourier-transform infrared spectroscopy (FTIR) by the Vertex 70v in transmission mode between 4000 cm −1 and 500 cm −1 , powder X-ray diffraction (PXRD) by the V8 Advance from Bruker from 35 to 80 degree, the Hitachi 4800 field emission scanning electron microscopy (FESEM) at the scale of 300 nm and the Euromex OX.2253 PLPHF fluorescence microscopy under the blue light mode with 1 s of exposure time.

RESULTS
The Au seed and grown Au NPs performed in the wine red while the seed was darker for its high concentration. Besides, both colloidal solutions were surveyed for the maximum absorbance wavelength by UV-Vis spectroscopy. They showed up at 520 nm for the seed and 521 nm for the 40 min synthesis nanoparticles in Figure 2. A transmission mode of FTIR was used with each modified step's outcome, including O 2 plasma, APTES, SA, and 11-MUA treatment. The range of wavelengths from 4000 cm −1 to 500 cm −1 was observed to record the feature spectrum peaks. Figure 3 shows the FTIR spectra from the measurement of the bonding interaction's oscillation on the silica surface. The crystallite structure and particle shape of the Au NPs coating surface were examined by the XRD analysis in the angle range from 35 o to 80 o and the FESEM at the scale of 300 nm. In Figure 4a, four peaks presented to four orientations were recorded at about 38, 43, 62, and 78 o, which was fully described in the Joint Committee's database on Powder Diffraction Standards, USA (JCPDS no. 00-004-0784) 16 . The Alexa Fluor 488 stained CRP anti-mouse IgG2a incubated over 5 h on two modified types of glass substrate were observed under a white light mode considered as the blank in Figure 5a-5c, and 1 s exposed blue light mode as the verification of coupling bio-factor appearance in Figure 5b-5d. In one hand of Figure 5ab, the model bonding procedure by SA was performed with fewer and fewer radiation markers localized by the green dots and streaks in which also told an uneven distribution of molecules. On the contrary, a moister, brighter, and denser behavior showed up on the Au NPs inclusion surface via the comparison captures in Figure 5 c-d without changing the biological incubation and fluorescence analysis. An optical system based on the T-mode (transmission mode) described in the previous study 17 was used for fluorescence detection and transform into an electric signal. The enhancement factor (EF) of the Au NPs coated the (1) equation calculated substrates in the mentioned reference. Figure 6 illustrates the fluorescence intensity and enhancement factor recorded from the CRP@Alexa 488 bonding on the comparison coating and non-coating substrate with six different concentrations listed in Table 1.

DISCUSSION
Due to the Mie theory 18 about the effects of nanoparticle size on its optical property, the recorded surface plasmon absorbance spectrum at about 520 nm in the UV-Vis range was coincidental to other studies about the LSPR application in biosensor 19 . Furthermore, the described color was presented to the nanoparticles' size in the range between 20 -40 nm, which was pointed out from other researches 20 and fit with the UV-Vis result.
Four repaired samples had the Si-O-Si asymmetric vibration at around 1060 cm −1 in common since the silica substrate 21 . The broadband at the range of 3485 cm −1 of Figure 3a is related to the silanol groups' stretching isolated vibration for being treated by O 2 plasma 22 . In the case of APTES treatment, the primary amine groups' stretching mode may be noticed at the band of 3362 cm −1 . Therefore, its bending and wagging mode also from the N-H bonding was shown up at 1562 and 759 cm −1, respectively, in Figure 3b Figure 3 d were assigned to the alkynes bonding from the MUA substances structure, which were the asymmetric and symmetric stretching modes. The familiar peaks are either APTES and SA spectrum but single and weaker since the shorter carbon chains 25,26 . Otherwise, the nanospheres showed up on the coated after being affected by the microscopy electron beam.
In detail, the face-centered cubic (FCC) structure of pure crystalline gold has appeared for the Bragg reflection. The (111) orientation got the strongest intensity of all, while the other recorded three, including (200), (220), and (311) had an approximately equal intensity. However, the most promising signal over the different immersion times was in the 16 h Au NPs coating surface, although the spectrum underwent a trend of increase. By using the Scherrer equation in calculating the crystalline size 27 after the spectra of the (111) orientation, the size is more or less 21.5 nm.   It also coincided with the FESEM captures considered by the nanoparticle's uniformity and the surface arrangement. By comparison with the nanoscale, the sphere's size is between 20 nm and 40 nm and consistent with the other analysis as UV-Vis and XRD. An similar trend of the increase happened in both methods, but a sharper shape can be mentioned by the Au NPs coating one. The no Au coating samples give the power under 0.4 µW while the coated reaches the peaks from 1 to 1.5 µW. Figure 6b presents the enhancement factor caused by the LSPR effect as desired, and the average 4.8 fold multiplication has occurred. The maximum enhancement resulted is in 0.01 mg/mL binding antibody and dropped slightly when it comes to 0.05 which the experimental manip- ulation may cause. It almost remained throughout the 10 times higher and more concentration that may be responsible for the limited carboxyl modified groups in the chamber work area. The LOD for this detection experiment can be accounted for 0.01 mg/mL, which is 100 times lower than the standard of CRP low-risk announcement at 1 mg/L (published in 2003 by The American Heart Association and U.S. Centers for Disease Control and Prevention), and much higher than the earlier research based on lossy-mode resonance (LMR) fiber devices 28 . The coefficient of variation (CV) was shown in Table 1 after 4 times repeat from each sample, demonstrating the repeatability and ideal if under 0.5%. The 0.05 mg/mL sample had a higher CV at 0.7%, which coincided with the lower EF unexpectedly.

CONCLUSIONS
The gold nanoparticles synthesized from the seedmediate method was proved the strong LSPR effect on the enhancement of the fluorescence signal. Besides, a simple and effective modification protocol was performed aimed to coat Au NPs and to bond the biofactor. The accomplished substrate pattern was built prepared for sensing through the curtain analysis of FTIR, XRD, FESEM, and fluorescence microscopy. As expected, a high sensitivity optical biosensor based on LSPR was designed and fabricated that got the remarkable EF at 4.8 folds with a low CV at 0.7%. There is an auspicious prospect in the ability of other noble metals utilization, switching into others structure or size, and a combination of various optical phenomena for the applications.