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Original Article
ARTICLE IN PRESS
doi:
10.25259/AJC_1097_2025

Analyzing duplex formation with abdominal aortic aneurysm biomarker on palladium nanocube attached interdigitated electrodes

, , , , , ,
aDepartment of Vascular Surgery, Affiliated Hospital of Yunnan University, Kunming, China
bDepartment of Medicine, Affiliated Hospital of Yunnan University, Kunming, China
cDepartment of Urology Surgery, Affiliated Hospital of Yunnan University, Kunming, China
dDepartment of Community Medicine, Saveetha Medical College & Hospital, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India
eInstitute of Nano Electronic Engineering, Universiti Malaysia Perlis (UniMAP), Kangar, Perlis, Malaysia
fFaculty of Chemical Engineering & Technology, Universiti Malaysia Perlis (UniMAP), Arau, Perlis, Malaysia
gDepartment of Electrical and Electronics Engineering, Dogus University, Umraniye, Istanbul, Türkiye
hDepartment of Technical Sciences, Western Caspian University, Baku AZ 1075, Azerbaijan
iDepartment of Oral and Craniofacial Sciences, Faculty of Dentistry, Universiti Malaya, Kuala Lumpur, Malaysia

*Corresponding authors: E-mail addresses: 13078760231du@sina.com (Lingjuan Du); subash@unimap.edu.my (S.C.B. Gopinath)

Received: , Accepted: ,
© 2026 Arabian Journal of Chemistry - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

Abdominal aortic aneurysm (AAA) is an inflated region in the lower part of body’s major artery (the aorta). Traditional screening methods of imaging are expensive, and researchers focus on alternative methods of biomarker screening with AAA detection. This research developed a method for duplex detection of two AAA biomarkers, namely, insulin like growth factor 1 (IGF1) and IGF binding proteins 1 (IGFBP1) for diagnosing AAA on palladium nanocube-modified interdigitated electrode (IDE) sensor. The probed surface with anti-IGFBP1 monoclonal antibody and anti-IGF1 aptamer was created on aminated palladium and performed the sensing with anti-IGF1 and anti-IGFBP1 antibodies. Palladium improved the probe attachment with aptamer-antibody on IDE surface and significantly enhanced the interactions of IGF1 and IGFBP1. The probe modified surface detects IGF1 and IGFBP1 as low as 10 fg/mL and 10 pg/mL with the R2 values of 0.9183 [y = 0.1105x2 – 0.2323x + 0.2181 R2 = 0.9975], and 0.9032 [y = 0.1123x2 – 0.2893x + 0.236 R2 = 0.9953], respectively, on a linear regression. Further, control performances with closely related biomarkers namely C-reactive protein (CRP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), and complementary aptamer sequence did not enhance the current and confirm specific detections IGF1 and IGFBP1. In addition, there is a current increment that was noted in IGF1 and IGFBP1 spiked serum samples without any interferences display the selective detection.

Keywords

Biosensor
IGF1
IGFBP1
Inflated aorta
Nanomaterial

1. Introduction

Abdominal aortic aneurysm (AAA) is an enlargement/bulge in the portion of aorta that extends through the abdominal aortal. This bulge weakens the aorta wall and makes it difficult to manage the pressure of pumped blood from the heart and difficult to handle the flood-flow. As a result, the weakening wall of aorta expands outward and enlarges, and grows continuously over time. Various factors such as tobacco, aging and some medical conditions weaken the aortas wall. When the aneurysm grows larger, the possibility is to burst and leads the life-threatening internal bleeding [1]. AAA size larger than 5.5 cm is particularly at risk, and no symptoms until they are close to rupture or ruptured. So that monitoring the size and growth of AAA before it exceeds 3 cm [2]. Since AAA is asymptomatic, it is difficult to identify until the point of rupture, which has a higher mortality rate [3]. This early screening programs for AAA was introduced in some countries like United Kingdom at the of age 65 and over, which drastically decreased the prevalence of AAA rupture. Conventional screening and evaluation of AAA using imaging methods such as magnetic resonance imaging, and CT angiogram. Even though these imaging methods have been shown to be effective, the expense of these programs can place a heavy financial strain on health care systems. So that, there is necessity of constant study for generating alternate approaches in diagnosing AAA.

Blood-based biomarkers show greater interest various disease diagnosis, including in the aneurysm field, and useful for earlier detection of AAA and monitoring its progression [4-6]. Moreover, these biomarkers help to measure the rate of expansion and size of aneurysm and also predict the risk of rupture [7]. Circulating insulin like growth factor 1 (IGF1) proves to be potential and plays a major role in AAA pathogenesis [8]. The liver produced IGF1 has various biological functions, which includes cell-growth, cell-migration, cell-survival and differentiation. IGF1 binds its receptor and is modulated by IGF binding proteins (IGFBPs). Higher levels of IGF1 and the increment of IGF1 and IGFBPs ratio are highly linked to AAA, in particular IGFBP1 is closely related to an increment of aortic diameter [9,10]. In addition, the level of IGF1 in serum is highly correlated with size and growth rate of AAA and predicts the necessity of surgery during AAA treatment. Besides, the recombinant therapy with IGF1 decreases the protein oxidation and proteolysis, which are increased significantly in AAA diseased tissues. Research identified that higher IGFBP-1 levels are observed in AAA tissues and thrombus, and they correlate the aneurysm size, indicating that it may be a suitable biomarker and contributes to AAA pathophysiology [11]. In addition, IGFBP-1 might promote platelet aggregation brought by IGF1, which could aid in the development of AAA. So that, quantifying these two markers helps to diagnose, prevent and monitor AAA and its conditions. This research was developed a nanomaterial mediated duplex detection of IGF1 and IGFBP1 on the probed (aptamer and antibody) surface of palladium nanocube-modified interdigitated electrodes (IDEs).

Nanomaterial applications in the biosensing field are a fast-growing direction, and researchers are now investigating various nanomaterials and their characteristics for the purpose of using in improving sensitivity in biosensors [12-14]. Nanomaterials have distinct electrical, physical, and mechanical properties, and are successfully adoptted in various sensing systems to improve the biosensing [15,16]. Due to the fast electron transfer, size, and higher surface area, materials such as gold, silver, platinum, rhodium, and palladium have been utilized to immobilize biomolecules on the electrodes and enhance the electric flow [17-19]. Recently different dimensioned materials show a greater attention for various biomedical applications including the bio-diagnosis [20-22]. Zero-three dimensioned particles are typically nanomaterials, having unique optical and electrical properties that make them suitable for applications, including electrochemical sensing and other electronic potentials [23,24]. In this research, palladium nanocube is used for the surface functionalization and to create AAA biosensor on IDE sensor. Palladium has been used as ionic conducting material and often utilized for electrolysis due to its higher catalytic activity. Since palladium has higher electrical conductivity, it is possible to enhance the electrical performances in the sensor, herein authors utilized palladium nanocube to immobilize anti-IGF1 aptamer and anti-IGFBP1 antibody on IDE sensor for quantifying IGF1 and IGFBP1. To demonstrate the high-performance detection of the above biomarkers, two probed surfaces were developed with antibody and aptamer attached on the palladium and modified on IDE though amine-linker. Aptamer is an artificial antibody generated from the randomized pool of molecules by using ‘SELEX’ method [25-27]. The high affinity aptamers help to detect the targets with higher selectivity and sensitivity [28-30]. Anti-IGF1 aptamer and anti-IGFBP1 antibody were used as capture probes to detect IGF1 and IGFBP1. Initially, the amine-linker was used to attach the anti-IGFBP1 polyclonal antibody on palladium and then the remaining space was filled with anti-IGF1 aptamer. Since antibody is larger, it can create the gap after binding on palladium, and the gaps were occupied by aptamer. The aptamer and antibody modified palladium attracts both IGF1 and IGFBP1, which were detected by interacting with anti-IGF1 and anti-IGFBP1 antibodies. This method of detection quantifies IGF1 and IGFBP1 with dual probes and monitor AAA and its severity.

2. Materials and Methods

2.1. Chemicals and biomolecules

Anti-IGF1 antibody, IGF1, IGFBP1, and anti-IGFBP1 antibody were received from Sino Biological, China. PEG-COOH and (3-Aminopropyl)triethoxysilane) (APTMS) were sourced from Sigma Aldrich, USA. EDTA, citric acid, and ethylene glycol were ordered from Thermo Scientific, USA. A Keithley 2450 (Voltammeter) was used to measure current voltage (I-V), with the aid of Kickstart software and a probe station. Anti-IGF1 aptamer was commercially synthesized by a local supplier. Aptamer: 5’-COOH-C6-ATACGGGAGCCAACACCAGATGCGAGGACGGTGGGTGGGAGGGTGGAGGTCTCGAGAGCAGGTGTGACGGAT-3’ and the complementary aptamer: 5’-COOH-C6-TATGCCCTCGGTTGTGGTCTACGCTCCTGCCACCCACCCTCCCACCTCCAGAGCTCTCGTCCACACTGCCTA-3’ [31]. The interdigitated electrodes (IDE) were purchased from Metrohm, Malaysia. The electrode surface was verified as imaging under 3D-nanoprofiler. Based on our earlier optimization studies, current-volt measurements provide a reliable output at an active range between 0 and 2 V.

2.2. Anti-IGFPB1 optimization on palladium-modified IDE

The palladium nanocubes were chemically synthesized from the precursor by following the method outlined previously [32]. The generated palladium nanocubes were verified by observing under scanning electron microscope and atomic force microscopy. Antibody and aptamer on palladium-modified IDE was prepared through the amine-linker. For this process, amine was created on palladium by using APTMS. Briefly palladium (1 mg/mL) was mixed in 2% of (3-aminopropyl)trimethoxysilane (APTMS) (10 mL in diluted in ethanol) and rested it for 2 h, after that the amine modified palladium (palladium-amine) recovered by centrifugation. For the probe preparation, IGFBP1 antibody was immobilized on palladium-amine. At first, IDE was hydroxylated by immersing electrode surface in KOH (5 mL of 1%) for 10 min and then 5 µL of palladium-amine was added and on the surface of the electrode rested for 2 h followed by rinsing the surface with 10 uL of ethanol. On the palladium-amine modified surfaces, 5 µL of anti-IGFBP1 antibody (62 nM) was introduced and kept for 30 min and then removed the unbound antibody by rinsing the electrode with 10 uL of 10 mM phosphate-buffered saline (PBS) (pH 7.4). The current was recorded for all the surface functionalization process. Similarly, the antibody concentrations at 125 nM, 250 nM, and 500 nM were immobilized on the palladium, and the current responses were recorded.

2.3. Anti-IGF1 aptamer optimization on palladium modified surface

On the antibody modified palladium, 5 µL of COOH-ended anti-IGF1 aptamer was attached. Aptamer concentration at 125 pM (5 µL) was added on the antibody-modified palladium and kept for 30 min. The current was recorded for the binding of aptamer on palladium surface. Similarly, the other aptamer concentrations of 250 pM, 500 pM, and 1 nM were tested on the palladium-antibody, and the current responses were recorded.

2.4. Duplex detection of IGF1 and IGFPB1

On the palladium-antibody-aptamer immobilized IDE, IGF1 and IGFBP1 were detected with its antibody. Sandwich pattern of antibody-IGFBP1-antibody, and aptamer-IGF1-antibody were performed. For the IGF1 detection, IGF1 with the concentration of 1 fg/mL (5 µL) was introduced on the surface and 200 nM of anti-IGF1 antibody was added. After rinsed with PBS (10 µL), the surface current was recorded. Similarly other concentrations of IGF1 (5 µL; 10 fg/mL, to 1 ng/mL) were detected. For IGFBP1 detection, IGFBP1 with the concentration of 1 pg/mL (5 µL) was introduced on the probe-modified IDE and then 200 nM of anti-IGFBP1 monoclonal (5 µL) interacted. After washing the surface with PBS (10 µL), the current was recorded. Similarly other concentrations of IGFBP1 (10 pg/mL to 1 µg/mL) were detected.

2.5. Specific, selective and stable detection of IGF1 and IGFPB1

To mimic the real-life detection of biomolecules, IGF1 and IGFBP1 were separately diluted in human serum (1:100 dilution; 5 µL) and dropped on aptamer-antibody modified palladium surfaces and then sandwiched with antibodies for IGF1 and IGFBP1. After washing the surface with PBS, the current responses were recorded for all the concentrations of IGF1 and IGFBP1. For control performance, five different experiments were conducted. (i) IGF1aptamer-IGF1-IGFBP1 antibody; (ii) IGFB1 antibody-IGFBP1-IGF1 antibody; (iii) IGF1 complementary aptamer sequence-IGF1-IGF1 antibody; (iv) IGF1 aptamer-CRP-IGF1 antibody; (v) IGFBP1 antibody-NTproBNP-IGFBP1 antibody. 5 µL solution was used for each experiment and 10 µL of PBS was used to wash after each immobilization step. The current responses for all the controls were compared with the current level of the specific target.

3. Results and Discussion

AAA biosensor was generated here by developing dual-probe surface with anti-IGF1 aptamer and anti-IGFBP1 antibody on palladium nanocube. Figure 1(a) shows the schematic illustration for AAA biosensor on IDE. Palladium was attached on the sensing surface by using the amine linker and then anti-IGFBP1 antibody was attached. Further, aptamer-ended with COOH was added on the surface to cover the excess surface on palladium. On these aptamers modified surfaces, IGF1 aptamer-IGF1-IGF1 antibody and Polyclonal IGFBP1 antibody-IGFBP1-monoclonal IGFBP1 antibody was performed to detect IGF1 and IGFBP1. With probe preparation, since antibody size is larger, it creates gap between the antibodies on palladium. In general, polyethylene glycol, Bovine serum albumin, and ethanolamine were used as blocking molecules after the probe immobilization, otherwise it will attract other biomolecules electrostatically and lead to false-positive results [33]. In this experiment, instead of blocking agent, author used IGF1-aptamer to cover the excess space on palladium. Apart from that palladium helps to attach the highest number of aptamer and antibodies on IDE and increases the detection performance. Usually, IDE sensors work well different sensing set-ups including, current-volt, electrochemical impedance and cyclovoltammetry. Among these, current-volt is a simpler and well fit with a dipole moment mechanism. In which, there are ionic changes between two electrodes (positive and negative) upon alterations with surface modification or molecular interaction on a sensing surface. In the current study, the dual-probed surface with aptamer, antibody on IDE can detect both IGF1 and IGFBP1. These two biomarkers (IGF1 and IGFBP1) quantifications help to identify and monitor the conditions associated with AAA.

(a) Schematic illustration of AAA biosensor on palladium-modified IDE (b) 3D-nanoprofiler image of IDE (Figure inset displays the original IDE).
Figure 1. (a) Schematic illustration of AAA biosensor on palladium-modified IDE (b) 3D-nanoprofiler image of IDE (Figure inset displays the original IDE).

3.1. Surface characterization

For the surface characterizations, three different microscopic observations were followed. To observe the surface intactness of IDE, it was observed under 3D-nanoprofiler and a clear difference between electrodes and gaps was noticed (Figure 1b). Figure inset displays the size and comparison of the obtained IDE. The synthesized palladium nanocubes were observed under scanning microscope (Figure 2a) and atomic force microscope (Figure 2b). From both observations, it is clear that the obtained palladium is in uniform size and shape and the nanocubes distributed uniformly. The measured size is calculated to be 90±10 nm.

Characterization of palladium. (a) Using scanning electron microscopy (SEM) (b) Using atomic force microscopy (AFM). Both images display the intactness and uniformity of nanocube generated.
Figure 2. Characterization of palladium. (a) Using scanning electron microscopy (SEM) (b) Using atomic force microscopy (AFM). Both images display the intactness and uniformity of nanocube generated.

3.2. Palladium-antibody immobilization on IDE

Anti-IGFBP1 antibody attachment on IDE was performed through palladium-amine. The process of antibody immobilization was monitored by using the changes in current volt. Figure 3(a) shows the current-volt graph of anti-IGFBP1 immobilization on palladium. The hydroxylated IDE displays the current as 1.88 E-07 A, it changes to 1.73 E-06 A after modified the surface with amine-palladium. This change of responses indicates the surface was modified with amine-palladium. Antibody was further added on palladium modified surfaces. When introduced 62 nM of anti-IGFBP1, current increment was noted from 1.73 to 2.54 E-06 A, which shows the attachment of antibody on palladium. Further, with increasing the antibody levels, the response of current also increased (Figure 3b). This interaction happened through COOH group in the antibody and the amine on palladium. In general, antibodies were directly attached to amine-modified surfaces through electrostatic interaction, but this leads to the improper attachment of antibodies on the electrode surface and leads to less interaction with the target molecule [34]. In addition, the stability and amount of antibody immobilization are also questionable. This study utilized palladium for the attachment of anti-IGFBP1 antibody, which improves immobilization with proper arrangement on IDE. The current response of antibody immobilization was saturated from 500 nM. After 500 nM of antibodies, there are no significant changes in current responses that were recorded and there is a slight current change noted between 250 and 500 nM. This study developed a probe with antibody and aptamer on palladium, and only a small space needed for aptamer immobilization. So that, 250 nM of antibody was found to be suitable concentration to create a probe surface for the detection of both IGF1 and IGFBP1.

Probe attachment on IDE. (a) Current-volt graph of anti-IGFBP1 antibody immobilization on palladium. Clear changes of current increment were noted with all antibody concentrations. (b) Current response of antibody attachment on palladium. Increase the antibody levels, the response of current also increased. The current response of antibody immobilization was saturated from 500 nM. (c) Current-volt graph of aptamer immobilization on palladium. Clear changes of current increment were noted with all aptamer concentrations. (d) Current response of aptamer attachment on palladium. Increasing the aptamer levels, increased the response of current levels. The current response of aptamer immobilization was saturated from 500 pM.
Figure 3. Probe attachment on IDE. (a) Current-volt graph of anti-IGFBP1 antibody immobilization on palladium. Clear changes of current increment were noted with all antibody concentrations. (b) Current response of antibody attachment on palladium. Increase the antibody levels, the response of current also increased. The current response of antibody immobilization was saturated from 500 nM. (c) Current-volt graph of aptamer immobilization on palladium. Clear changes of current increment were noted with all aptamer concentrations. (d) Current response of aptamer attachment on palladium. Increasing the aptamer levels, increased the response of current levels. The current response of aptamer immobilization was saturated from 500 pM.

3.3. Preparation of aptamer-antibody modified palladium

On the antibody modified palladium, aptamer was dropped to fill the space between antibodies. For this, aptamer concentrations from 125 pM to 1 nM was introduced on IDE- palladium-antibody modified electrode surface and the current changes were recorded for each concentration. As shown in Figure 3(c), 125 pM of aptamer on palladium shows the current changes from 6.57 to 9.25 E-06 A, which confirms the attachment of aptamer molecules on palladium. Further, with increasing the concentration of aptamer, the increments in current responses were noted and saturated from 500 pM. There is no significant response in current increment was noted after 500 pM, which means that 500 pM of aptamer is enough to cover the free space on palladium (Figure 3d). Since aptamer is smaller in size, it can fit the space between antibodies and create the uniform probe surface for the detection of IGF1 and IGFBP1. Apart from that, palladium makes more stable form of aptamers and antibodies on IDE, which helps to bind higher number of IGF1 and IGFBP1. Previously, researchers developed a dual probe to quantify the biomarker Aβ peptides for the diagnosis of Alzheimer disease (AD). They utilized gold nanourchin to create dual probes with aptamer and antibody. On the gold urchin, the antibody and aptamer for AβO were attached and detected Aβ as low as 10 fM. AβO can simultaneously binds with aptamer and antibody and increases the current and lowered the detection limit [35]. In the current experiment, authors prepared the antibody and aptamer on palladium for different target molecules (IGF1 and IGFBP1), which can detect both biomarkers in the blood sample and help to diagnose AAA and the associated conditions.

3.4. Detection of IGF1 by palladium-antibody-aptamer modified IDE

IGF1 was quantified on palladium-antibody-aptamer modified IDE. The surface immobilized anti-IGF1 aptamer was interacted with IGF1 and sandwiched by anti-IGF1 antibody. Figure 4(a) shows the current level of different IGF1 concentrations binding with its probe. After the aptamer immobilization, the excess sensing surfaces were blocked with a blocking agent (PEG-COOH) and reduced the nonspecific binding. After that, IGF1 followed by IGFBP1 antibody was added to create a sandwich pattern of aptamer-IGF1-antibody. 1 fg/mL of IGF1 increased the current from 2.82 to 4.31 E-05 A, which shows IGF1 interaction with the probe. Further, when the concentration of IGF1 was increased, level of current also increased (Figure 4b). This current increment indicates the detection of IGF1 with appropriate probe. The difference in current was plotted in an excel sheet and calculated the detection limit of IGF1 is 10 fg/mL with an R-squared value of 0.9528 (Figure 4c). Previous research used to detect IGF1 with its specific aptamer and IGFBP1. Since IGF1 can interact with IGFBP1, they made the sandwich assay of aptamer-IGF1-IGFBP1 to detect the IGF1. Iron oxide nanoworm was utilized to immobilize the aptamer on the electrode. This sandwich assay lowers the detection of IGF1 to 1 fM [36].

Detection of IGF1. (a) Current level of different IGF1 binding with its aptamer and antibody. Clear changes of current increment were noted with all IGF1 concentrations. (b) Current response of different IGF1 concentrations by its aptamer and antibody. The concentrations of IGF1 increased the level of current. (c) Limit of detection of IGF1. The difference in current was plotted in an excel sheet and calculated the detection limit of IGF1 at 10 fg/mL with the squared value of 0.9528.
Figure 4. Detection of IGF1. (a) Current level of different IGF1 binding with its aptamer and antibody. Clear changes of current increment were noted with all IGF1 concentrations. (b) Current response of different IGF1 concentrations by its aptamer and antibody. The concentrations of IGF1 increased the level of current. (c) Limit of detection of IGF1. The difference in current was plotted in an excel sheet and calculated the detection limit of IGF1 at 10 fg/mL with the squared value of 0.9528.

3.5. Detection of IGFBP1 by palladium-antibody-aptamer modified IDE

After the IGF1 detection, the same probe modified surface was used to quantify the IGFBP1. The surface immobilized anti-IGFBP1 polyclonal antibody was interacted with IGFBP1 and sandwiched with anti-IGFBP1 monoclonal antibody. Figure 5(a) shows the current response of different IGFBP1 interaction with its poly and monoclonal antibodies. 1 pg/mL of IGF1 increased the current from 2.82 to 3.67 E-05 A, which confirms the IGF1 interaction with its aptamer. Further, when the concentration of IGFPB1 was increased, level of current also increased (Figure 5b). This current increment indicates the detection of IGFBP1 with and antibody. The difference of current was plotted in an excel sheet and calculated the detection limit as 10 pg/mL with an R-squared value of 0.933 (Figure 5c). Since the IGF1 and IGFBP1 concentration play a major role for the size of AAA, these two biomarkers quantify to identify the condition of AAA. When comparing these two-detection systems, IGF1 shows the higher response of current than IGFBP1 detection (Figures 4 and 5). The increment of current response depends on various factors, which includes the surface functionalization, and binding affinity of biomolecules. In the case of IGF1, an aptamer was used as the capturing probe, while IGBP1 antibody was used in IGFBP detection. Since the aptamer binding affinity with target is higher than antibody, it can attract a higher number of molecules on the electrode and lower the detection limit [37,38]. So that, IGF1 has higher current responses than IGFBP1. At the same time, higher number of IGFBP1 antibody was achieved through the palladium, which lowers the detection limit of IGFBP to 1 pg/mL.

Detection of IGFB1. (a) Current level of IGFBP1 binding with its poly and monoclonal antibodies. Clear changes of current increment were noted with all IGFBP1 concentrations. (b) Current response of different IGFBP1 concentrations by its poly- and monoclonal-antibodies. When the concentrations of IGFBP1 were increased the levels of current also increased. (c) Limit of detection of IGFBP1. The difference of current was plotted in an excel sheet and calculated the detection limit of IGFBP1 as 10 pg/mL with the squared value of 0.933.
Figure 5. Detection of IGFB1. (a) Current level of IGFBP1 binding with its poly and monoclonal antibodies. Clear changes of current increment were noted with all IGFBP1 concentrations. (b) Current response of different IGFBP1 concentrations by its poly- and monoclonal-antibodies. When the concentrations of IGFBP1 were increased the levels of current also increased. (c) Limit of detection of IGFBP1. The difference of current was plotted in an excel sheet and calculated the detection limit of IGFBP1 as 10 pg/mL with the squared value of 0.933.

3.6. Detection of IGF1 and IGFBP1 in spiked serum samples

To mimic the real-life sample detection for AAA diagnosis, both IGF1 and IGFBP1 was diluted in human serum and added on the palladium-modified IDE and sandwiched with IGF1 and IGFBP1 antibodies. The increment of current was noted in both targets by increasing the IGF1 and IGFBP1 concentrations without any interference (Figures 6a and b). This was achieved through the specific interaction of IGF1 aptamer with IGF1 and IGFBP1 antibody with the IGFBP1. Research has proved that the level of IGF1 in serum is highly correlated with the size of AAA and helps to predict the need for surgery. Also, IGFBP1 was higher significantly in larger AAA than control patients. Further, IGFBP1 was found in the luminal region of AAA thrombus, and it is higher in media conditioned by AAA thrombus than in media layer and healthy media. The level of IGFBP1 in control is 497 pg/mL, which was significantly increased to 834 pg/mL in patients with larger AAA [11]. So that, the quantification of IGF1 and IGFBP1 in serum samples predict the AAA size and also identifies the need of surgery with AAA patients. The serum samples contain the heavy molecular weight protein such as albumin, globulin and other clotting proteins, which interfere the detection of IGF1 and IGFBP1 in human serum. Since our sensor is highly sensitive with target molecule, it detects IGF1 and IGFBP1 in the serum without any interference and increases the current responses in all IGF1 and IGFBP1 concentrations, which confirms the selective detection of target molecules.

Detection of IGF1 and IGFBP1 in spiked serum samples. To mimic the real-life sample detection for AAA diagnosis, both (a) IGF1 and (b) IGFBP1 were diluted in human serum and added on the palladium-modified IDE and sandwiched by IGF1 and IGFBP1 antibodies. The increment of current was noted in both targets by increasing IGF1 and IGFBP1 concentrations without any interference. (c) Control performances with various combinations of control proteins and complementary aptamer: (1) IGF1-aptamer-IGF1-IGFBP1 antibody; (2) IGFB1 antibody-IGFBP1-IGF1 antibody; (3) IGF1 complementary aptamer-IGF1-IGF1 antibody; (4) IGF1 aptamer-CRP-IGF1 antibody; (5) IGFBP1 antibody-NTproBNP-IGFBP1 antibody; (6) Specificity with IGF1; (7) Specificity with IGFBP1. There is no noticeable current recorded in control experiments, which confirms the specific detection of IGF1 and IGFBP1.
Figure 6. Detection of IGF1 and IGFBP1 in spiked serum samples. To mimic the real-life sample detection for AAA diagnosis, both (a) IGF1 and (b) IGFBP1 were diluted in human serum and added on the palladium-modified IDE and sandwiched by IGF1 and IGFBP1 antibodies. The increment of current was noted in both targets by increasing IGF1 and IGFBP1 concentrations without any interference. (c) Control performances with various combinations of control proteins and complementary aptamer: (1) IGF1-aptamer-IGF1-IGFBP1 antibody; (2) IGFB1 antibody-IGFBP1-IGF1 antibody; (3) IGF1 complementary aptamer-IGF1-IGF1 antibody; (4) IGF1 aptamer-CRP-IGF1 antibody; (5) IGFBP1 antibody-NTproBNP-IGFBP1 antibody; (6) Specificity with IGF1; (7) Specificity with IGFBP1. There is no noticeable current recorded in control experiments, which confirms the specific detection of IGF1 and IGFBP1.

3.7. Control performances for specific detection of IGF1 and IGFBP1

Control performances with various combinations of control proteins and complementary aptamer sequence were conducted. Five different experiments were conducted. (i) IGF1-aptamer-IGF1-IGFBP1 antibody; IGFB1 antibody-IGFBP1-IGF1 antibody; IGF1complementary aptamer-IGF1-IGF1 antibody; IGF1 aptamer-CRP-IGF1 antibody; IGFBP1 antibody-NTproBNP-IGFBP1 antibody. CRP and NTproBNP are also biomarkers for AAA, which was used as the control proteins. As shown in Figure 6(c), there is no noticeable current recorded in control experiments, which confirms the specific detection of IGF1 and IGFBP1. In this surface functionalization PEG-COOH was used to block the excess amine surfaces, which usually causes the attraction through the electrostatic interaction. Research has proved that amine modified silica surface non-specifically attracts gold nanomaterial conjugated streptavidin, and it was completely avoided by the PEG-based polymer [28]. In addition, PEG based blocking agents provides the better biomolecular attachment on the electrode surface, which increases the chances of probe binding with target molecules. Herein, PEG-COOH covers the excess amine on palladium after attaching aptamers and antibodies, which increased the chance of specific detection of IGF1 and IGFBP1.

4. Conclusions

Abdominal aortic aneurysms (AAA) develop in the aorta wall and, if untreated, cause a major blood conduit to dilate extensively and ultimately rupture. Low survival rates and high mortality rates for aortic rupture survivors are two negative outcomes. Early identification and monitoring the size of AAA is mandatory to provide a better treatment before rupturing. In this study, IGF1 and IGFBP1 sensing systems were developed on palladium-modified IDE to diagnose AAA. Palladium was attached on IDE through amine-linker and then anti-IGF1 aptamer and anti-IGFBP1 antibody were attached on amine-modified palladium to create a dual probe for the detection of IGF1 and IGFBP1. On the surface of palladium aptamer and antibody were attached as dual probes. Higher numbers of antibody and aptamer were immobilized on IDE through palladium nanomaterial. A linear range IGF1of 10-15 to 10-9 and 10-12 to 10-6 of IGFBP1 were tested on a probe modified surface, which shows higher increment in current responses with increasing concentrations. The detection limit of IGF1 and IGFBP1 lowered to 10 fg/mL and 10 pg/mL with R2 values of 0.9183, and 0.9032, respectively. Further, IGF1 or IGFBP1 spiked serum samples increase the current with increasing concentrations of IGF1 and IGFBP1 without interference, confirms the selective detection. In addition, control experiments with relevant proteins, namely CRP, NTproBNP and complementary aptamer sequence did not show current changes, indicating the specific detection of IGF1 and IGFBP1. This palladium-modified probe molecule detects IGF1 and IGFBP1 at its lower levels and diagnose the severity of AAA.

Acknowledgement

Funded by the Association foundation program of Yunnan provincial science and technology department and Kunming medical university [202401AY070001-021].

CRediT authorship contribution statement

Guojian Li: Data curation, formal analysis, investigation, resources, writing original draft preparation, Jiankun Chen and Xi Guo: Data curation, methodology, writing-reviewing and editing, Thangavel Lakshmipriya: Data curation, validation, visualization, writing-reviewing and editing, Subash C.B. Gopinath: Conceptualization, formal analysis, supervision, visualization, resources, writing-reviewing and editing, Yeng Chen: Validation, visualization, writing-reviewing and editing, Lingjuan Du: Conceptualization, formal analysis, project administration, supervision, resources, 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.

Data Availability

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References

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