Improved immunoassay for Insulin-like Growth Factor 1 detection by aminated silica nanoparticle in ELISA

Journal Pre-proof Improved Immunoassay for Insulin-like growth factor 1 Detection by Aminated Silica Nanoparticle in ELISA Xin Hong (Conceptualization) (Methodology) (Data curation) (Investigation) (Validation)Writing-original draft)Writing-reviewing and editing), Xingyu Hong (Conceptualization) (Data curation) (Investigation) (Validation)Writing-reviewing and editing), Haomin Zhao (Methodology) (Data curation) (Investigation) (Validation)Writing-reviewing and editing), Chong Chen (Data curation) (Validation)Writing-reviewing and editing), Subash C.B. Gopinath (Validation)Writing-original draft)Writing-reviewing and editing), Daniel Lim (Software) (Data curation)Writing-reviewing and editing), Yeng Chen (Data curation)Writing-reviewing and editing), Guangjun Yan (Conceptualization) (Investigation) (Validation) (Visualization) (Supervision)Writing-reviewing and editing)

PII:

S1359-5113(19)31468-0

DOI:

https://doi.org/10.1016/j.procbio.2019.12.019

Reference:

PRBI 11881

To appear in:

Process Biochemistry

Received Date:

2 October 2019

Revised Date:

9 December 2019

Accepted Date:

24 December 2019

Please cite this article as: Hong X, Hong X, Zhao H, Chen C, Gopinath SCB, Lim D, Chen Y, Yan G, Improved Immunoassay for Insulin-like growth factor 1 Detection by Aminated Silica Nanoparticle in ELISA, Process Biochemistry (2019), doi: https://doi.org/10.1016/j.procbio.2019.12.019

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Improved Immunoassay for Insulin-like growth factor 1 Detection by Aminated Silica Nanoparticle in ELISA

Xin Hong1, Xingyu Hong1, Haomin Zhao1, Chong Chen1, Subash C.B. Gopinath2,3, Daniel Lim4, Yeng Chen5, Guangjun Yan1,*

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Department of Vascular Surgery, China-Japan Union Hospital of Jilin University, ChangChun, Jilin, 130031, China 2 School of Bioprocess Engineering, Universiti Malaysia Perlis, 02600 Arau, 3Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000 Kangar, Perlis, Malaysia. 4 Department of Oral & Craniofacial Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. 5 Department of Oral & Maxillofacial Clinical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia.

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Xin Hong: [email protected] Xingyu Hong: [email protected] Haomin Zhao: [email protected] Chong Chen: [email protected] Subash C.B. Gopinath: [email protected] Daniel Lim: [email protected] Yeng Chen: [email protected] Guangjun Yan: [email protected]

*Correspondence to:[email protected]

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Graphical abstract

Improved ELISA

Selectivity

IGF1+ IGF1+ C IGF2 IGFBP3 IGF1

Conventional ELISA Sensitivity

Substrate

Secondary antibody HRP

OH/CO

APTES-SiO2 KOH surface

SiO2

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ELISA plate

Size distribution

70 60 50 40 30 20 10 0

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IGF1

Scattered light Intensity (a.u.)

Anti-IGF1

Highlights

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Abdominal aortic aneurysm (AAA) is the vascular disease forming when aorta swells Insulin-like Growth Factor 1 (IGF1) is the circulating biomarker for AAA detected Improved ELISA through the aminated silica nanoparticle (SiO2) mediation 400pM of IGF1 detected on amine-SiO2 modified ELISA & 12.5nM in conventional ELISA Detected in the mixture of IGF1 and relevant proteins (IGF2 and IGFBP3)

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100

Particle size (nm)

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Abstract

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Abdominal aortic aneurysm (AAA) is the vascular disease forming when aorta in the abdomen swells. AAA with the size of 5.5 and above has possibility to rupture and leads a death. Insulin-like Growth Factor 1 (IGF1) is the circulating biomarker for AAA diagnosis, in this study IGF1 was detected by the improved Enzyme linked Immunoassay (ELISA). The conventional ELISA necessitates the improvement due to the lesser sensitivity than modern sensors. Higher biomolecular immobilization is one of the wise ways to enhance the sensitivity. This study was aimed to capture a higher number of IGF1 on ELISA surface through the aminated silica (SiO2) nanoparticle with the size measured to be 80-100 nm. On the amine-SiO2 modified surface, Glutaraldehyde (GLU) complexed IGF1 was chemically bonded. It was found that 400 pM of IGF1 detected on amine-SiO2 modified ELISA, whereas the conventional ELISA without nanoparticle mediation shows the limit of detection as 12.5 nM with ~30 times lower detection. The selectivity experiment was carried out with the mixture of IGF1 and relevant proteins (IGF2 and IGFBP3), and it was noticed that anti-IGF1 selectively detected IGF1. This nanoparticle-mediated surface immobilization captured a higher rate of antigen on a larger surface created for highperformance detection.

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Keywords: Immunodetection; Silica nanoparticle; Abdominal aortic aneurysm; Insulin-like Growth Factor 1; Vascular biomarker

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1. Introduction

Abdominal aortic aneurysm (AAA), a vascular disease has been noticed when the

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size of the aorta become bigger in abdomen area due to the unusual swelling [1]. Smoking and high blood pressure are found to be the primary reasons for AAA [2].

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AAA region with the diameter 5.5 and above will have possibility to break and cause

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the excess bleeding, and it can be the fatal [3]. This abnormal complication makes the situation to identify AAA before being attained the severe stage. IGF1 is a protein with MW 21 kDa, found to be one of the suitable biomarker for AAA [4]. In this study, IGF1 was quantified towards improving Enzyme linked Immunoassay (ELISA) at its lower level. This improvement is aimed for complementing the currently available potential sensing strategies.

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Improved biosensor is mandatory to elevate the diagnosing system and extend the human lifespan. Various sensors and sensing techniques have been demonstrated to detect the diseases on the potential sensing surfaces [5]. Among the reported sensing strategies, ELISA is one of the promising assays of detection from small molecule to the whole cell. Due to the positive features such as lower detection limit, cheaper, easier to use, and, reproducibility, until now several diseases have been

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screened by ELISA [6]. In the ELISA, basically the aimed target is immobilized on the polystyrene (PS) substrate and then a suitable antibody is added. Followed by the secondary antibody conjugated enzyme such as, horseradish peroxidase is

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permitted to interact with primary antibody. Finally, the interaction is monitored by the suitable substrate [7]. The limit of detection on ELISA plate is highly depending on

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the target or antibody immobilization on PS plate. Biomolecules can attach on PS

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plate through the physical adsorption by the hydrophobic group of the biomolecules. But this might cause the weak and improper binding and leads to the lower detection limit. Focusing on the proper immobilization of protein or antibody on PS is an

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efficient way and it may improve the detection of the target. Different chemical, electrostatic, and physical methods have been used to immobilize the antibody or

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protein on PS plate. The pre-mixture of (3-aminopropyl)triethoxysilane (APTES) and

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antibody binding on the ELISA plate was shown to improve the detection by ~55 times than the conventional ELISA [8]. Previously, Lakhmipriya et al. used the gold nanoparticle to immobilize clotting protein factor IX on ELISA plate and enhanced the detection limit to 100 pM [9]. Also, chemical conjugation of HIV-p24 protein was carried out through the amine-aldehyde link on ELISA and improved the detection to

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1 nM [10]. In this work, amine modified silica nanoparticle was used to immobilize IGF1 on ELISA surface to diagnose AAA. Nanoparticle application in the field of sensing field is widely spread due to its high stability with biomolecular conjugation and providing a higher sensitivity in detection. Different nanoparticles such as gold, silver, graphene, silica, platinum, titanium and copper were used in sensing purpose in order to lift the sensing strategy [11–14]. Among a range of nanoparticles, silica nanoparticle (SiO2) has its unique feature of

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low-toxicity, optically transparent and bioinert. Most interestingly, the surface of the silica was functionalized easily with the well-established silane technology [15]. This study was utilized SiO2 nanoparticle to immobilize the detection of IGF1 on ELISA. It

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is a challenging process, especially with lower molecular weight proteins like IGF1. The introduction of SiO2 nanoparticle is enhanced the performance of immunoassay

2. Materials and Methods

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in ELISA by accommodating the higher amount of IGF1.

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2.1. Reagents and biomolecules

(3-Aminopropyl)triethoxysilane (APTES) and Phosphate buffered saline (PBS) with pH 7.4 were purchased from Sigma-Aldrich (USA), Anti-mouse-IgG-conjugated

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horseradish peroxidase was received from Thermo-Scientific (USA), ELISA 5X

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coating buffer was purchased from BioLegend (UK), Horseradish Peroxidase (HRP) substrate and Bovine serum albumin (BSA) were received from Promega (USA), The ELISA plate was procured from Becton Dickinson (France), The analytical ELISA reader was from Fisher Scientific (Malaysia), Human IGF1, IGF2, IGFBP3 and antiIGF1 were from Sino Biological, China. 2.2. Characterization of SiO2 nanoparticle

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The Field Emission Scanning Electron Microscope (FESEM, GEMINI) was used to analyse the morphology of SiO2 nanoparticle. The FESEM/EDX spectrum was studied to found the compositional elements in the SiO2 nanoparticle. UV-visible spectroscopy was scanned (200-350 nm) to analyse the peak maximum. Moreover, particle size analyser was performed to analyse the size of the SiO2 nanoparticle. 2.3. Optimization of anti-IGF1 and secondary antibody immobilization Before detecting IGF1, initially the suitable dilution of anti-IGF1 to be used was

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optimized. For that, anti-IGF1 with the dilutions of 1:250, 1:500, 1:1000 and 1:2000 (in 1x coating buffer) was added on different wells in PS plate and kept at room temperature (RT) for overnight. Next day, the remaining PS plate surface was

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blocked by 3% BSA (diluted in PBS buffer) for 1 h at RT. After the surface was

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washed by PBS five times, the anti-IGF1 (1:1000 dilution from 1 mg/mL) was interacted for 3 hr, followed by secondary antibody conjugated HRP (1:1000 dilution

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from 1 mg/mL) was added and kept 1 at RT. Between each step the surface was washed thoroughly by washing buffer (PBS with 0.05% of Tween-20). Finally, the

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interaction of primary and secondary antibody was monitored by adding 50 µL of the substrate for HRP. The absorbance wavelength was read at 405 nm by UV-Vis

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Spectrometry. Control experiment was carried out without secondary antibody

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interaction.

2.4. Optimization of APTES-SiO2 conjugates on ELISA surface

To optimize the suitable concentration for the efficient chemical bonding of APTES and SiO2, different combinations of APTES and SiO2 were mixed and immobilized on the ELISA plate. Six different combinations such as 0.5% APTES mixed with 1 or 2 6

mg/mL of SiO2; 1% APTES mixed with 1 or 2 mg/mL of SiO2, 2% APTES mixed with 1 or 2 mg/mL of SiO2. These mixtures were individually added on ELISA plate and kept 3 h at RT. The surface was washed with PBS buffer and 1:1000 dilution of antiIGF1 was added. And then the remaining surfaces were blocked by BSA. Finally, 1:1000 dilution of secondary antibody was added. These interactions are analysed with substrate for HRP.

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2.5. Optimization of GLU-IGF1 conjugates on ELISA surface

To improve the detection, GLU linker was mixed with IGF1 and dropped on APTES-

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SiO2 modified ELISA surface. GLU with the concentrations 0.25, 0.5, 1 and 2% (diluted in PBS) were mixed independently with 100 nM of IGF1 and kept at RT for

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30 min. These mixtures was added on APTES-SiO2 modified ELISA surface and

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kept for 1 h at RT. And then 1:1000 dilution of primary antibody followed by 1:1000 dilution of secondary antibody-HRP were interacted. Before the antibody attachment, the surface was blocked by BSA to avoid any fouling from biomolecules. Finally, the

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substrate was added to find the interaction. The absorbance wavelength was

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recorded at 405 nm.

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2.6. Stability assay on SiO2-antibody modified ELISA surface To check the stability of improved ELISA surface, APTES-SiO2 modified plate was kept at 4°C. IGF1 was detected on this plate at the time intervals of 1, 2, 3 and 4 weeks. The same experimental procedures were followed as described above.

2.7. Comparison of limit of detection between conventional ELISA and improved ELISA by SiO2 7

Limit of detection of IGF1 in the improved ELISA system was compared with the conventional ELISA. Experiments followed as described. Improved ELISA: (i) Mixture of APTES-SiO2 was added on ELISA surface; (ii) IGF1 concentrations from 12.5 pM to 100 nM were mixed with 1% of GLU were added on different wells; (iii) Blocking by 3% BSA; (iv) anti-IGF1 with 1:1000 dilution was added; (v) secondary antibody-HRP with 1:1000 dilution was added; (vi) substrate for HRP was added.

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Conventional ELISA: (i) IGF1 concentrations from 12.5 pM to 100 nM in 1X coating buffer were added on ELISA surface; (ii) Blocking by 3% BSA; (iii) anti-IGF1 with 1:1000 dilution was added; (iv) secondary antibody-HRP with 1:1000 dilution was

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added; (v) substrate for HRP was added.

2.8. Selective detection of IGF1

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Three different control experiments were performed to confirm detection of IGF1.

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Control 1 (C-1): without primary antibody: Control 2 (C-2): without secondary antibody; Control 3 (C-3): without primary and secondary antibody. Moreover,

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Selective detection of IGF1 was performed by mixing of IGF1 with other proteins namely, IGF2 or IGFBP. A 3 nM of IGF1 was mixed with 3 nM of IGF2 or IGFBP3

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and dropped on APTES-SiO2 modified ELISA surface, in the presence of GLU. Then blocking solution, primary antibody and secondary antibody were added with the

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proper time interval as stated above. Substrate for HRP was added to find the specific interaction of IGF1 with its antibody. Error bar indicates the averaged values from triplicates (n=3). p value with <0.05 was considered to be significant.

3. Results and Discussion

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Abdominal aortic aneurysm (AAA) is defined as the swelling of the aorta, especially in the abdomen area. It generally happens at the older age of >60, smoking and blood pressure were found to be the primary causative for AAA [16]. IGF1 is the circulating biomarker for AAA, in the current study IGF1 was detected by ELISA. Improvement in the ELISA was performed by IGF1 immobilization on ELISA surface through

APTES-conjugated

SiO2

nanoparticle.

Higher

number

of

IGF1

immobilization was created; obviously leads to the lower detection limit. Figure 1 is

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the schematic representation for improving ELISA by APTES-SiO2. Initially, the ELISA surface was hydrolysed by 1% of KOH, it was proved that the hydrolysed surface with KOH improved the binding of APTES [8]. The premixed APTES-SiO2

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were added on KOH-modified surface and then GLU-IGF1 was added. Anti-IGF1 followed by secondary antibody-HRP was added on this surface. Finally, the

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substrate for HRP was added to read the colour changes. SiO2 nanoparticle gives

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the opportunity to bind a greater number of antigens on the sensing surface. APTESmodified SiO2 attract GLU-IGF1 effectively, lead to the lower detection limit. Moreover, nanoparticle modified biomolecules displays higher stability and

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accommodate higher number of biomolecules, which improves the detection system [17–19]. In this research, high affinity IGF1 detection with improved ELISA surface

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was compared with the conventional ELISA.

3.1. Morphological and structural analyses of SiO2 nanoparticle Figure 2a & b show FESEM image and EDX spectra of SiO2 nanoparticle. From these results, it was noticed that SiO2 nanoparticle has the spherical shape with the smooth surface and the diameter was found to be 80 to 100 nm. EDX spectra clearly indicate the presence of SiO2 nanoparticle. The particle size analyse confirms the

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size of the nanoparticle within the range ~100 nm (Figure 2c). Figure 2d displays the UV-Visible spectroscopy analysis on the SiO2 nanoparticle, is showing the peak maximum around 230 nm.

3.2. Anti-IGF1 and secondary antibody interaction on ELISA surface Before detect IGF1, initially, the suitable concentration of anti-IGF1 was determined. The excess or lower antibodies influence the detection with IGF1. As shown in figure

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3a, 1:250 dilution of anti-IGF1 did not show the significant difference in absorbance from the control experiment. With increasing the dilution to 1:500, the absorbance was increased. Inset image in the figure shows the colour changes and it was clearly

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observed from 1:500 dilution. The dilution 1:1000 and 1:2000 shows the saturated absorbance, and the optimized dilution factor 1:1000 of anti-IGF1 was used for

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further experiment.

3.3. Identifying suitable APTES and SiO2 concentration

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After anti-IGF1 optimization, it is necessary to optimize the suitable combination of APTES and SiO2 conjugates. In general, APTES molecule easily attracts other

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biomolecules or nanoparticle through the electrostatic or physical adsorption. Lakshmipriya et al. have found that streptavidin-conjugated gold nanoparticle was

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physically bind on the APTES-modified surface and polyethylene glycol-based polymer was used to cover the APTES surface to avoid biofouling [20]. To optimize the suitable combination of APTES, and SiO2, six different combinations of these molecules binding was tried on the ELISA plate. As shown in figure 3b, 0.5% APTES with 1 or 2 mg/mL of SiO2 shows the less absorbance, at the same time 1% and 2% APTES with 1 or 2 mg/mL of SiO2 display the similar level of absorbance. But, in 10

control experiments APTES with 2% shows a minor increment in the absorbance, indicating the nonspecific binding. The inset photo image clearly shows colour changes with APTES 1% with 1 mg/mL of SiO2. In conclusion, 1% APTES with 1 mg/mL of SiO2 combination can be used for further experiments. 3.4. Improving detection by GLU conjugated IGF1 To improve the IGF1 detection, IGF1 was premixed with GLU as the linker. GLU has two aldehydes at its both ends, one side can interact with amine of IGF1, and the

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other side can binds the amine from APTES. Previously, it was proved that the premixed GLU and protein immobilization on ELISA surface improved the detection limit [10]. To optimize the suitable Glu concentration in this study, GLU with different

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concentrations were mixed with the similar 100 nM IGF1 and added on the APTESSiO2 modified ELISA surface. It was found that 0.25% of GLU shows the slight

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change of color with the lower absorbance. From 0.5%, the absorbance started to

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increase, and at 1 and 2%, it reached its saturation point (Figure 4a). From this obtained date it was concluded that the optimized level at 1% GLU will be ideal for

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further experiments and used.

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3.5. Stability of antibody-SiO2 modified ELISA surface Before go for the detection of IGF1, the stability assay on modified ELISA surface

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was carried out with different time intervals. As shown in figure 4b, 100 nM of IGF1 detection on APTES-SiO2 modified ELISA surface shows the similar absorbance, indicates the immobilized APTES-SiO2 is stable enough on ELISA surface. This stability was similar until 3 weeks and a slight reduction was found from the week 4.

3.6. Comparison of limit of detection between IGF1 by conventional ELISA and improved ELISA using SiO2 11

Detection of IGF1 on improved ELISA surface was compared with the conventional ELISA (unmodified). IGF1 concentrations from 12.5 pM to 100 nM were detected on both surfaces and the absorbance of each concentration was compared (Figure 5a&b). It was noticed that in the conventional ELISA until 6 nM, there is no significant change with the absorbance was noted. However, from 12.5 nM of IGF1, the absorbance started to increase. In the case of modified ELISA plate, an apparent

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change in absorbance was noted from 400 pM. Moreover, in all the concentrations of IGF1, modified ELISA plate shows the higher range of absorbance than the conventional ELISA, this might be due to the higher and proper immobilization of IGF1 on ELISA surface through APTES-SiO2. The limit of detection with modified

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ELISA was calculated to be 400 pM, while it was found as 12.5 nM in the

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conventional ELISA, this is ~30 times improvement in the detection.

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3.7. Selective detection of IGF1 on modified APTES-SiO2 ELISA surface Three different control experiments namely, control 1 (C-1): without primary

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antibody: Control 2 (C-2): without secondary antibody; Control 3 (C-3): without primary and secondary antibodies were evaluated. As shown in figure 6a, all the

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control experiments were did not show the significant changes in the absorbance, at the same time with IGF1, it clearly increased the absorbance, indicating the specific

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detection of IGF1 by the modified ELISA strategy. Moreover, selective detection of IGF1 was carried out with premixed IGF1 with different related proteins and detected on APTES-SiO2 ELISA surface. A 1 nM of IGF1 was mixed with IGF2 or the receptor protein IGFBP3 and immobilized on the APTES-SiO2 ELISA surface. It was found that, even in the mixed sample, anti-IGF1 was clearly detected the IGF1, indicating the selective detection of IGF1 by the modified ELISA surface (Figure 6b). 12

4. Conclusion Abdominal aortic aneurysm (AAA) is belonging to the vascular disease family, localized by the swollen area of the abdomen aorta. IGF1 is the circulating biomarker for AAA, herein IGF1 was detected on (3-Aminopropyl)triethoxysilane (APTES)-silica nanoparticle (SiO2) modified ELISA surface. APTES (1%) was mixed with SiO2 (1 mg/mL) found as the ideal condition for the immobilization. The limit of detection of

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IGF1 was detected as low as 400 pM on the improved ELISA plate, while it was 12.5 nM in the conventional ELISA. This is ~30 times increment in the improvement for the detection. Moreover, in different control experiments, IGF1 can only bind with

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anti-IGF1 antibody indicating the specific detection and also selective experiment with premixed proteins, anti-IGF1 could only recognise the IGF1, indicating the

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selective detection. This improved ELISA technique helps to diagnose AAA at an

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advanced level, can be followed on other sensing surfaces. Since this method used the chemical linker to immobilize the protein, the strategy can also be utilized for

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other molecules biomolecules such as DNA, RNA and peptide.

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CRediT author statement

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Xin Hong: Conceptualization, Methodology, Data curation, Investigation, Validation, Writing original draft preparation, Writing-Reviewing and Editing Xingyu Hong: Conceptualization, Data curation, Investigation, Validation, WritingReviewing and Editing Haomin Zhao: Methodology, Data curation, Investigation, Validation, WritingReviewing and Editing Chong Chen: Data curation, Validation, Writing-Reviewing and Editing Subash C.B. Gopinath: Validation, Writing original draft preparation, WritingReviewing and Editing Daniel Lim: Software, Data curation, Writing-Reviewing and Editing Yeng Chen: Data curation, Writing-Reviewing and Editing 13

Guangjun Yan: Conceptualization, Supervision Writing-Reviewing and Editing

Investigation,

Validation,

Visualization,

Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1:

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Figure legends

Schematic representation IGF1 detection on APTES-SiO2 modified ELISA. GLU-IGF1 was added on APTES-SiO2 immobilized surface.

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Anti-IGF1 was allowed to interact IGF1 followed by secondary antibodyHRP was added. These interactions were monitored by the substrate for

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HRP.

Characterization of SiO2. (a) scanning electron microscopy; (b) energy-

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Figure 2:

dispersive

X-ray

(c)

Particle

size

analyser;

(d)

UV-visible

spectrophotometry. Size was measured to be 80-100 nm.

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Figure 3: Optimization of anti-IGF1 dilution. Anti-IGF1 with the dilutions of 1:250,

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1:500, 1:1000 and 1:2000 were tested to interact the secondary antibody. 1:1000 was found as the optimum. (b) Optimization of APTES and SiO2. Different combinations of APTES were mixed with SiO2 and tested on ELISA. APTES with 1% mixed with 1 mg/mL of SiO2 was

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found as the optimum. Figure inset displays the diagrammatic representation. Error bar indicates the averaged values from triplicates

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(n=3) with the standard deviations are in the range of ± 0.005 to 0.01. p

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value was significant with 0.035.

Figure 4: (a) Optimization of GLU concentration. GLU with the concentrations of 0.25, 0.5, 1 and 2 mg/mL were mixed independently with 100 nM of IGF1 and tested on ELISA. GLU with 1 mg/mL was found as the optimum. (b) Stability assay for improved ELISA was modified by APTES-SiO2. APTES-SiO2 on ELISA was kept for different intervals and tested the detection of IGF1 at 100 nM. All the wells showed the similar 17

detection levels are indicating the strong stability of APTES-SiO2 on ELISA. Figure inset displays the diagrammatic representation. Error bar indicates the averaged values from triplicates (n=3) with the standard deviations are in the range of ± 0.005 to 0.01. p value was significant

Figure 5:

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ro of

with 0.04.

Comparative analysis. (a) Dose-dependent IGF1 interaction with anti(b)

re

IGF1 on ELISA in the presence and absence of nanoparticle.

Absorbance measurement by UV-Vis spectrometer. Measured at 405

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na

lP

nm. Figure inset displays the diagrammatic representation.

Figure 6: Specificity and Selectivity analyses. (a) Specific detection of IGF1 on APTES-SiO2 modified ELISA. Three different control experiments were performed. Control 1 (C-1): no primary antibody: Control 2 (C-2): no secondary antibody; Control 3 (C-3): no primary and secondary antibodies. The absorbance was found with only IGF1 indicating the 18

specific detection. (b) Selective detection of IGF1 on APTES-SiO2 modified ELISA. 1 nM of IGF1 was mixed with IGF2 or IGFBP3 and added on APTES-SiO2 mediated ELISA. In the mixed samples, anti-IGF 1 was clearly detected IGF1, indicating the selective detection. Error bar indicates the averaged values from triplicates (n=3) with the standard deviations are in the range of ± 0.005 to 0.01. p value was significant

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lP

re

-p

ro of

with 0.045.

19