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Zinc oxide-gold mediates surface-enhancement in Enzyme-linked immunosorbent assay for high-sensitive diagnosing acute pancreatitis
* Corresponding author: E-mail address: yangjinxinxin@sina.com (J. You)
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Received: ,
Accepted: ,
Abstract
Acute pancreatitis is the condition of an inflammation in the pancreas, occurring due to trauma, abuse, and alcohol consumption. The level of amylase in the blood is as an established marker, regularly looked by physicians for diagnosing pancreatitis. Enzyme-linked immunosorbent assay (ELISA) can be a ‘gold standard’ method to quantify the α-amylase level in the biological fluids. Improving ELISA is mandatory to enhance the detection of α-amylase, herein, zinc oxide (ZnO) nanomaterial modified ELISA-surface was introduced to detect the level of α-amylase. ZnO was immobilized on the ELISA well through alkalinization followed by 3-Aminopropyltriethoxysilane (APTES)-linkage. Anti-α-amylase monoclonal antibody was added on ZnO-modified ELISA well. On the modified surfaces, amylase was sandwiched using mono- and polyclonal-antibodies conjugated gold nanoparticles. This nanomaterials-modified ELISA detects amylase as low as 0.6 ng/mL on a linear regression with 0.5 to 40 ng/mL in the buffer [y = 0.0797x + 0.0849; R2 = 0.9746]. The amylase was also detected from the saliva samples without any interference, indicates the specificity of method generated. This modified ELISA quantifies a lower level of α-amylase and helps to diagnose acute pancreatitis and the associated issues.
Keywords
α-amylase
Acute pancreatitis
ELISA
Gold
Zinc oxide
1. Introduction
Acute pancreatitis (AP) is the condition of pancreas inflammation due to the obstructive duct and alcoholism, which leads to nausea, pain, fever, and even death in some severe cases [1,2]. AP is a common reason for the hospitalization among gastrointestinal related issues in America. Based on the progression, AP was categorised into 3 levels, which include mild, moderate, and severe [3,4]. It is necessary to identify AP at its mild stage to provide a better treatment and speedy-recovery. The pancreas has two major functions namely secreting sugar regulating hormone and excretes the digestive enzymes. Lipase, α-amylase, protease, glycoprotein, and inflammatory cytokines are found as the suitable biomarkers for diagnosing AP [5-7]. The abnormal level of the pancreatic enzymes can be considered for AP biomarker analyses. Among others, amylase is a well-established biomarker and often utilized by physicians to monitor the condition of AP [8]. The normal level of α-amylase in the blood serum ranges between 36 and 128 mg/dL, while it elevates several times greater than the normal range during the inflammation, which indicates pancreatitis [9]. Different analytical methods, such as colorimetric assay, spectrophotometry, enzyme-linked immunosorbent assay (ELISA), turbidimetry, and liquid chromatography have been used to quantify the level of α-amylase [10]. Among them, ELISA is commonly used method to identify the level of α-amylase due to its unique features, such as simple, easy-to-use, sensitive, specific, and lower-cost. Researchers are still working towards improving the ELISA and its sensitivity for detecting α-amylase at lower level to differentiate the inflamed pancreas from a normal condition.
ELISA is a heterogeneous immunoassay used to analyse and screen the clinical samples for diagnosing diseases [11,12]. In the conventional ELISA, the poly/monoclonal antibody is immobilized on the ELISA polystyrene (PS) surface through the electrostatic interaction and then the biological sample containing antigen is allowed to bind with the immobilized antibody. After rinsing the well, the specific antibody (poly/mono) was added to the well to make a sandwich assay of antibody-antigen-antibody. Further, enzyme conjugated secondary antibody interacted with antibody and then the substrate for enzyme is added. The absorbance changes indicates the quantity of antigen in the sample [13,14]. Instead of antibody, aptamer is also used in ELISA for detection target molecule. Aptamer is an artificial antibody, which can be possibly be used as a capture molecule in ELISA for detecting the target. Since aptamer is a smaller molecule, attaching on ELISA well is challenging. A chemical, physical or nanomaterial as be used to link or attach the aptamer on the PS as well and then follow the sandwich pattern which can be done with aptamer-target-antibody. In this work, zinc oxide (ZnO) was utilized to improve anti-amylase antibody immobilization on ELISA polystyrene plate (PS). In general, surface functionalization with primary antibody or antigen plays a vital role for improving the sensitivity [15]. However, it is still challenging to immobilize the smaller-sized molecules, such as peptide, nucleic acid, aptamer, and protein on PS well. Various methods were carried out by researchers to improve the immobilization process on ELISA as well [16]. Among them, nanomaterial-mediated biomolecular immobilization has been proven to improve the sensitivity and selectivity of the antigen in ELISA [17-19].
Nanomaterials are nanosized materials effectively applied in various biomedical applications [20,21]. Nanomaterials in ELISA significantly improves the sensitivity and ease of operation. In the conventional ELISA, lower enzyme-to-antibody ratio limits the sensitivity and controls the signal-to-noise ratio [22,23]. To improve the sensitivity, need to increase the number of enzymes in the final detection solution containing target-antibody-enzyme complex [23]. This will increase the signal amplification and lower the detection limit. For this process, metal oxide nanoparticles have been utilized for surface functionalization to increase the complex of antigen with antibody on the ELISA well [24]. In particular, the first biomolecular (antibody or antigen) immobilization plays a major role in improving the antigen-antibody complex on the ELISA well. Nanomaterials work as a link and thus improve the capture molecule immobilization on the ELISA well. If molecules directly attach to the ELISA well, they attach randomly without proper orientation, which reduces the chances of target attraction. When biomolecules are immobilized on nanomaterials, it increases their numbers and also aids in immobilizing on PS well with a proper orientation. Herein, ZnO and AuNP are utilized to capture a higher number of anti-amylase antibodies on the ELISA PS-well. ZnO was modified on PS well through an amine-linker, and then the antibody was attached to the amine-modified ZnO. Through this method higher number of antibody immobilization was achieved. Further, α-amylase was sandwiched with antibodies and then secondary antibody-horseradish peroxidase (HRP)-modified gold nanoparticle (GNP) was added. These interactions monitored by HRP substrate. Both ZnO and AuNP increases the antibody-antigen interaction on the ELISA well and improved the detection of α-amylase.
2. Materials and Methods
2.1. Reagents and biomolecules
Human saliva was obtained from BIOIVT, USA. α-amylase, gold nanoparticle (AuNP; 20 nm size), sodium hydroxide, ZnO, PEG-COOH (400), anti-amylase antibody, HRP, substrate for HRP, and ELISA plate were purchased from Sigma Aldrich, USA. ELISA reader was used from R & M Chemicals (U.K.). Antibody was conjugated with gold nanoparticle by amines, followed by carboxylic acid activation with 1-Ethyl-3-3-dimethylaminopropyl carbodiimide (EDC)-N-Hydroxysuccinimide (NHS) as described previously [25].
2.2. Sol-gel preparation of ZnO nanoparticle
The chemical wet synthesis method was used to synthesize ZnO nanoparticles using sodium hydroxide and zinc chloride. Briefly, zinc chloride (1 g) was prepared in 100 mL organic solvent and then 2 g of NaOH was added into the solution. The solution was continuously stirred at 100°C for 1 h and later the solution was kept at 80°C. The generated white colored solid was collected by centrifugation (15,000 x g for 15 min) and the solid portion was washed with water to remove the unused salt and base. The collected ZnO was dissolved in 100 mL of water for further use.
2.3. Amine modification on ZnO
The synthesized ZnO was modified with amine to attach the antibody on ELISA well. Briefly, 2% of 3-Aminopropyl triethoxysilane (APTES) (1 mL) was prepared by the mixing ethanol (93%) and water (5%) and then ZnO (1 mg/mL) was added into the APTES and placed in a hot plate for 2 h at 50°C. The amine-modified ZnO was separated by centrifugation (10000xg, 10 min) and then washed the modified ZnO with water to remove the excess APTMS and separated by centrifugation. Finally, APTMS-ZnO was dispersed in 25% ethanol for further ELISA surface functionalization. PS well was washed five times in between each step using 10 mM phosphate buffered saline (PBS) (pH 7.4) containing 0.05% of tween-20.
2.4. Comparison of antibody immobilization on ELISA well with and without ZnO
Antibody immobilization on ELISA well was compared with and without ZnO. Following steps are involved in this surface functionalization. Method 1 (Without ZnO): (i) Antibody with concentrations 100 to 1600 nM (diluted in PBS) were added in PS well and rested it for 2 h; (ii) 1% PEG-COOH was added and rested it for 30 min; (iii) Secondary antibody-HRP (1:1000 dilution) was introduced and rested for 30 min. Surface was rinsed with ELISA washing buffer and add the HRP substrate, absorbance was measured. Method 2 (With ZnO): (i) PS well was filled with 1% KOH (10 min) for hydroxylation process and rinsed with distilled water; (ii) APTMS-ZnO (1 mg/mL) was added and rested it for 1 h and washed the surface with 25% ethanol; (iii) Antibody at concentrations from 100 to 1600 nM were added in PS wells and rested for 30 min and washed with PBS buffer; (iv) PEG-COOH (1 mg/mL) was filled in the antibody modified well; (v) Secondary antibody-HRP was introduced and rested for 30 min. Surface was rinsed with ELISA washing buffer, the HRP substrate was added and the absorbance (at 450 nm) was measured.
2.5. Comparison of absorbance increment with and without AuNP conjugated secondary antibody
Secondary antibody with and without AuNP were optimized on the PS well. Following steps are involved in this surface functionalization: (i) PS well was filled with 1% KOH (10 min) for hydroxylation process; (ii) APTMS-ZnO (1 mg/mL) was added and rested it for 1 h and the surface was washed with 25% ethanol; (iii) Antibody with concentrations of 100 to 1600 nM were added in PS well and rested it for 30 min; (iv) PEG-COOH (1 mg/mL) was filled in the antibody modified well; (v) Secondary antibody-HRP (1:250 to 1:4000 dilutions) was added and rested for 30 min. Surface was rinsed with ELISA washing buffer, the HRP was added as substrate, and absorbance (at 450 nm) was measured. The same experiment was conducted with Secondary antibody-HRP+AuNP (1:250 to 1:4000 dilutions).
2.6. Comparison of α-amylase modified ELISA with conventional ELISA
Modified α-amylase ELISA with ZnO and AuNP were compared with conventional ELISA. Following steps are involved in the detection process. Conventional ELISA: (i) Antibody with concentration of 800 nM was added in PS well and rested it for 2 h; (ii) 1% PEG-COOH was added and rested it for 30 min; (iii) α-amylase levels from 0.6 to 40 ng/mL were diluted on PBS and then added in the PS well, and kept for 30 min; (iv) Polyclonal antibody (1:1000 dilution) was added and rested it for 30 min; (v) 1:1000 dilution of secondary antibody-HRP was added and rested it for 30 min. Surface was rinsed with ELISA washing buffer, the HRP substrate was added, and the absorbance was measured. Modified ELISA: : (i) PS well was filled with 1% KOH (10 min) for hydroxylation process; (ii) APTMS-ZnO (1 mg/mL) was added and rested it for 1 h and washed the surface with 25% ethanol; (iii) Antibody with the concentrations of 100 to 1600 nM were added in PS well and rested it for 30 min; (iv) PEG-COOH (1 mg/mL) was filled in the antibody modified well; (iv) α-amylase levels from 0.6 to 40 ng/mL were diluted in PBS and then added to the PS well, and kept for 30 min; (v) Polyclonal antibody (1:1000 dilution) was added and rested it for 30 min (vi) Secondary antibody-HRP (1: 250 to 1:4000 dilution) was introduced and kept for 30 min. Surface was rinsed with ELISA washing buffer, added the HRP substrate, and absorbance (at 450 nm) was measured.
2.7. Detection of α-amylase from saliva
To detect the α-amylase from saliva sample, saliva was purchased and used instead of α-amylase in the modified ELISA. All other experimental procedures followed as explained earlier. The limit of detection was considered the lowest concentration of an analyte on a calibration line against the background signal (S/N = 3:1; limit of detection (LOD) is a standard deviation of the baseline + 3σ.
3. Results and Discussion
Nanomaterial mediated ELISA was introduced to quantify the level of α-amylase for monitoring the condition of acute pancreatitis. ZnO and AuNP were utilized to enhance the performances of antibody-antigen-antibody interactions on the ELISA PS well. Figure 1 shows the graphical representation of nanomaterial modified α-amylase ELISA assay. PS well was hydroxylated with KOH and then APTMS-ZnO was modified on the well. Further, monoclonal antibody was allowed to bind with the immobilized ZnO through the amine linker, and then, the uncovered amine surface was covered with PEG-COOH. α-amylase was added and then sandwiched with polyclonal antibody. Secondary antibody-HRP-AuNP was linked with the monoclonal antibody and then HRP substrate was added to observe the color changes. With this surface functionalization ZnO was used as the linker to attach the monoclonal antibodies on the ELISA well, which playing a crucial role for the detection of α-amylase. When APTMS covered ZnO was attached on the surface, it attracts higher number of antibodies and increase the antibody immobilization with the proper orientation on the ELISA well. When antibody was directly attached on the ELISA well, it immobilizes randomly without proper orientation. ZnO helps to improve the antibody immobilization with the proper alignment, which helps to attract higher number of α-amylase. Further, secondary antibody was conjugated with AuNP increase the number of enzymes on the ELISA well, which helps to increase the enzyme level and lower the detection limit of α-amylase [26]. This was achieved through the multiple antibodies and enzymes on the surface of a single nanoparticle, which enhances the performance of ELISA, and this was compared with the conventional ELISA.
- Graphical representation of nanomaterial modified α-amylase ELISA. PS well was hydroxylated with KOH and then APTMS-ZnO was immobilized. Monoclonal antibody was allowed to bind the immobilized ZnO, and then uncovered amine-surface was masked by PEG-COOH. α-amylase was added and sandwiched by polyclonal antibody. Secondary antibody-HRP-AuNP was linked with the polyclonal antibody and then HRP substrate was added to see the color changes.
3.1. Antibody immobilization on ELISA well with and without ZnO
Primary antibody immobilization on the ELISA is an important step for the detection of antigen and diagnose the diseases [27]. In most ELISA methods, antigen first attached on the ELISA well, which was done through the electrostatic interaction on the polystyrene plate. This interaction is weaker and not enough to cover the complete surface with the targeted molecule. So that, researchers use various chemical interactions and linker to increase the molecular immobilization on the ELISA well [28-30]. In the current study [31], amine-modified ZnO was utilized as a linker to attach α-amylase antibodies and compared with the conventional method. Figure 2 shows comparison of primary antibody immobilization on ELISA well with and without ZnO. As shown in Figure 2(a), the antibody concentrations from 100 to 1600 nM were immobilized directly on the ELISA well. In which, concentrations at 100, and 200 nM shows the slight changes of absorbance and from 400 nM, and the absorbance was increased and saturated from 800 nM. In a second method, antibodies were attached on the ELISA well through ZnO, the absorbance was increased from 100 nM, and the increment was increased gradually. Color changes were also visible from 100 nM and increased the intensity with increasing antibody concentrations, indicating that higher number of antibodies was attached when ZnO was used as a linker. APTMS surrounding the ZnO acts very possible to attract the higher antibodies on the ELISA well and also ZnO provides the better arrangement of antibodies, which increase the antibody numbers on the ELISA. Since there is a slight difference of absorbance between 800, and 1600 nM, 800 nM was optimized for further α-amylase quantification.
- Primary antibody immobilization on the ELISA (a) Without ZnO; (b) With ZnO. ZnO modified surface increases the absorbance in all antibody concentrations and saturated from 800 nM. (Experiment was performed with 5 replicates and averaged).
3.2. Comparison of absorbance with and without AuNP conjugated secondary antibody
Secondary antibody conjugated enzyme immobilization is the last step and the crucial part of ELISA for signal amplification. The absorbance signal is highly depending on the number of enzymes in the final solution of ELISA, which is monitored with the suitable substrate. To enhance the enzyme immobilization, here we conjugated the secondary antibody-HRP with the AuNP and compared with the conventional ELISA method. Figure 3 shows the comparison of different dilution of secondary antibody-HRP immobilization (1: 250 to 1:4000 dilution) on ELISA well with and without AuNP. As shown in Figure 3(a), with 1:250, dilution, the absorption was increased from 0.048 O.D to 0.28 O.D, which clearly confirms the presence of secondary antibody-HRP on the ELISA well. Further increase the dilution, the absorbance also increased. It was noticed that, from 1:1000 dilution, drastic increment of absorbance was recorded, and then the absorbance was saturated. In the case of AuNP conjugated antibody, from 1:250 dilution, the absorbance was higher compare with the previous experiment, in particular from 1:1000 dilution, the absorbance was drastically increased compared with the previous experiment (Figure 3b). The same 1:1000 dilution of secondary antibody shows the absorbance of 0.48 and 0.56 O. D without and with AuNP. This may be due to the many numbers of attachment of antibodies on the single GNP and increase the enzyme immobilization on the ELISA well. The dilution from 1:1000 shows there is no significant increment of absorbance, and it also necessary to balance the amount of excess enzyme to reduce the signal to noise ratio, 1:1000 dilution of secondary antibody-HRP-AuNP conjugation was chosen for further α-amylase quantification.
- Secondary antibody-HRP optimization. (a) without AuNP; (b) With AuNP. Antibody with AuNP increases the absorbance with all antibody concentrations and saturated from 1:1000 dilution. Experiment was performed with 5 replicates and averaged.
3.3. Comparison detection of α-amylase with modified ELISA and conventional ELISA
α-amylase was detected by using both modified ELISA and conventional ELISA for comparison. For this process α-amylase concentration from 0.6 to 40 ng/mL was titrated and identified by the ELISA assay (Figure 4). Figure 4(a) shows the absorbance level of α-amylase detection with conventional ELISA. There is no absorbance and color changes in the 0.6 ng/mL of α-amylase, and with increasing the α-amylase to 1.2 ng/mL, a slight increment in absorbance and color changes were noticed. Further, increment with the α-amylase levels to 2.5, 5, 10, 20, and 40 ng/mL, the absorbances were increased to 0.18, 0.3, 0.42, 0.48, and 0.52 O.D., respectively. The limit of detection of α-amylase was 1.2 ng/mL with the conventional ELISA. In the modified ELISA, there is a clear difference in color changes were noticed from 0.6 ng/mL and when increased the α-amylase levels to 1.2, 2.5, 5, 10, 20, and 40 ng/mL, the absorbances were increased and with enhancing the intensity of color. With all concentrations of α-amylase, the absorbance was higher in the modified ELISA than the conventional ELISA (Figure 4b). The difference in absorbance value for each α-amylase concentration was plotted in an excel sheet. The limit of detection of α-amylase was calculated as 0.6 ng/mL with the modified ELISA at an R2 value of 0.9746, which is two times lower than the conventional ELISA (Figure 5a).
- Comparative detection of α-amylase. (a) Conventional ELISA; (b) Modified ELISA. In the modified ELISA, there is a clear difference of color changes were noticed from 0.6 ng/mL. With increasing α-amylase concentrations to 1.2, 2.5, 5, 10, 20, and 40 ng/mL, the absorbances were increased and the intensity of color increased. The color and absorbance changes were noticed from 1.2 ng/mL in the conventional ELISA. (Experiment was performed with 5 replicates and averaged).
- (a) Limit of detection of α-amylase. The difference in absorbance value for each α-amylase concentration was plotted in an excel sheet and the limit of detection of α-amylase was calculated as 0.6 ng/mL by the modified ELISA with an R2 value of 0.9746, which is two times lower than the conventional ELISA. (b) Detection of α-amylase in saliva sample. α-amylase was detected from a saliva sample by using the modified ELISA. A clear absorbance increment and current changes were noted in all samples, indicating that modified ELISA can identify α-amylase from the biological sample without any interferences. (Experiment was performed with 5 replicates and averaged).
3.4. Detection of α-amylase from saliva sample
α-amylase was detected from the saliva samples by using the modified ELISA. For this experiment, saliva was purchased and used instead of α-amylase in the modified ELISA. As shown in Figure 5(b), clear absorbance increment and current changes were noted in all the five samples, indicating that modified ELISA can identify the α-amylase from the biological sample without any interferences. In most of ELISA assays, bovine serum albumin (BSA) was used as a blocking agent to cover excess PS surface by electrostatically and reduce the biofouling. In the current surface functionalization, APTMS was used to attach ZnO on PS well, which easily attracts other biomolecules non-specifically. To reduce this issue, PEG-COOH was used as a blocking agent and binds with free amine surfaces and reduce nonspecific binding of biomolecules. Since saliva contains various biomolecules other than amylase, there is a possibility of increasing the signal due to the nonspecific binding. PEG-COOH reduces the nonspecific binding brings a specific binding of α-amylase with antibody. The present study provided a concreate method mediated by nanomaterial for diagnosing acute pancreatitis. The demonstrated sensing platform can be expanded with potential biomarkers that have been studied clinically [30-35].
4. Conclusions
Acute pancreatitis is an inflammation condition in the pancreas, it is a common reason for most of the gastrointestinal disorders. Identifying AP with a suitable biomarker is mandatory to follow-up the condition of pancreas. α-amylase is a well-established biomarker for diagnosing acute pancreatitis, herein, high-sensitive nanomaterial mediated ELISA was introduced to quantify the level of α-amylase. ZnO nanoparticle was utilized to attach the higher number of antibody immobilization on the ELISA well and secondary antibody conjugated AuNP improves the presence of enzymes on the ELISA well. It was proved that antibody attachment through ZnO was higher compared with the conventional method and the enzyme immobilization also increased when AuNP conjugated with secondary antibody. These modified ELISA identifies α-amylase as low as 0.6 ng/mL, which is two times lower than the conventional ELISA. Further, the modified ELISA tested with the saliva sample for α-amylase detection, it shows a clear absorbance and color changes, indicating the detection of α-amylase from saliva without any interferences. This modified ELISA identifies the α-amylase and helps to monitor the condition of acute pancreatitis.
Acknowledgment
Funded by Natural Science Foundation of Shaanxi Province [2018JM7122].
CRediT authorship contribution statement
Sheng Yu: Data curation, Formal analysis, investigation, writing original draft preparation, Thangavel Lakshmipriya: Formal analysis, methodology, writing-reviewing and editing, Subash C.B. Gopinath: Formal analysis, resources, validation, visualization, writing-reviewing and editing, Yuan Seng Wu and Yeng Chen: Formal analysis, writing-reviewing and editing, Jinzhi You: Conceptualization, formal analysis, project administration, resources, validation, visualization, writing-reviewing and editing. All are read and agreed with the content of manuscript.
Declaration of competing interest
There are no conflicts of interest.
Declaration of generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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