Gold‑Nanoparticle‑Enhanced ELISA and Interdigitated Electrodes for Ultra‑Sensitive Detection of Human Factor IX Deficiency

Abstract

Early, precise detection of low‑abundance biomarkers is pivotal for pre‑emptive treatment of clotting disorders. We present a robust surface‑functionalization strategy that combines pre‑alkaline polystyrene activation, amine (APTES) modification, and gold nanoparticle (GNP) conjugation to immobilize human factor IX (FIX) with unprecedented sensitivity. Optimized conditions—2.5 % APTES and 25 % of 15‑nm GNPs—yielded a limit of detection (LOD) of 100 pM in ELISA, a 60‑fold improvement over conventional protocols. In an interdigitated electrode (IDE) electrochemical platform, the same surface chemistry achieved a 25 pM LOD, allowing detection of FIX in 1:640 diluted human serum (≈125 pM). These findings demonstrate the versatility of GNP‑assisted surfaces across diverse sensing modalities and set a new benchmark for diagnosing coagulation deficiencies.

Introduction

Accurate, rapid diagnostics underpin modern medicine’s capacity to intervene before disease complications arise. Enzyme‑linked immunosorbent assays (ELISA) remain the gold standard for viral, bacterial, and protein biomarkers, yet their sensitivity is constrained by probe immobilization chemistry. Mis‑oriented antibodies, uneven coverage, and nonspecific adsorption can inflate limits of detection (LOD) by orders of magnitude. Recent advances harness the plasmonic properties of gold nanoparticles (GNPs) to enhance signal transduction and probe density. Here, we integrate GNPs into a chemically modified ELISA surface to improve FIX detection—a critical clotting factor whose deficiency leads to severe bleeding disorders. Complementary experiments on interdigitated electrodes (IDE) validate the approach across optical and electrochemical readouts.

Materials and Methods

Reagents and Biomolecules

APTES and human serum were from Sigma‑Aldrich. FIX protein and anti‑FIX antibody (Abcam, Malaysia); anti‑mouse‑IgG‑HRP (Thermo‑Scientific). ELISA plates (Becton Dickinson). GNPs of 10, 15, and 80 nm (Nanocs) were used for optimization.

Surface Preparation

Polystyrene (PS) plates were hydroxylated with 1 % KOH for 1 h, then incubated with 2.5 % APTES in ethanol for 5 h at room temperature. 25 % of 15‑nm GNPs were applied (or premixed with FIX) and incubated overnight at 4 °C.

Optimization of APTES and GNP Concentrations

We tested 1.25, 2.5, and 5 % APTES combined with 25, 50, and 100 % GNP dilutions. 250 nM FIX was introduced, followed by blocking (2 % BSA), primary (1:1000) and secondary (1:1000) antibodies, and HRP substrate. Optical density (OD) at 405 nm measured the signal. The 2.5 % APTES/25 % GNP condition provided the best signal‑to‑noise ratio.

Size Determination of GNPs

Using the optimal surface chemistry, 10, 15, and 80‑nm GNPs were evaluated. 15‑nm GNPs delivered comparable OD to 80‑nm but with superior colloidal stability, thus selected for subsequent experiments.

Specificity Controls

Four control configurations were assembled: (C1) FIX‑BSA‑anti‑mouse‑IgG‑substrate; (C2) FIX‑BSA‑FIX antibody‑substrate; (C3) BSA‑FIX antibody‑anti‑mouse‑IgG‑substrate; (S) FIX‑BSA‑FIX antibody‑anti‑mouse‑IgG‑substrate. Absence of signal in controls confirmed specific antigen–antibody interactions.

Comparative ELISA Sensitivity

Three ELISA formats were compared: (1) conventional; (2) APTES‑GNP‑FIX (FIX immobilized after GNP attachment); (3) APTES‑GNP premixed FIX (FIX and GNP mixed before surface binding). Each was tested across 0–200 nM FIX. The premixed method achieved an LOD of 100 pM; the direct GNP‑FIX method 200 pM; conventional ELISA 6 nM.

Human Serum Detection

Serum samples (20–1280 × dilution) were premixed with GNPs and applied to the modified surface. FIX was detected at 1:640 dilution (≈125 pM). Spiking experiments with 0.125–8 nM FIX into 1:640 serum confirmed linear response.

IDE Fabrication and Surface Modification

Standard photolithographic processes yielded 240 nm Al IDEs on 310 nm SiO₂. Surfaces were cleaned with 1 M KOH, then functionalized with 2.5 % APTES, followed by GNP–FIX premix. Blocking employed ethanolamine; anti‑FIX antibody detected the analyte. Current responses were recorded to determine LOD.

Results and Discussion

Pretreatment Efficacy

1 % KOH activation introduced hydroxyl groups, enhancing APTES attachment and subsequent GNP capture. Without this step, amine density—and thus GNP loading—was insufficient.

GNP Characterization

UV‑Vis spectroscopy showed a single plasmon peak at 550 nm, confirming monodispersity. TEM confirmed ~15 nm diameter. FTIR revealed amine signatures, and a zeta potential of –6.9 mV indicated stable colloids.

Surface Optimization

2.5 % APTES/25 % GNP provided optimal probe density while minimizing nonspecific adsorption. Higher APTES or GNP concentrations led to surface crowding and false positives.

Size Selection

15‑nm GNPs offered the best balance of surface area and colloidal stability, achieving similar OD to 80‑nm particles but with less aggregation.

Specificity Verification

Controls C1–C3 produced negligible OD, while the specific assay (S) yielded a robust colorimetric change, confirming that the signal arose from true FIX–antibody binding.

Enhanced Sensitivity

Figure 5 demonstrates the dramatic LOD improvement. The premixed GNP–FIX method allowed visual detection down to 200 pM, whereas conventional ELISA required >3 nM. The increased antigen density and plasmonic amplification underpin this performance boost.

Serum Application

FIX detection in 1:640 serum (125 pM) was achieved without interference from abundant serum proteins, underscoring the method’s practical relevance for clinical diagnostics.

IDE Complementation

Electrochemical detection using the same surface chemistry yielded an LOD of 25 pM, surpassing ELISA by fourfold. Serum dilution of 1:1280 produced a measurable current shift, validating the IDE platform for point‑of‑care testing.

Conclusion

The GNP‑assisted surface chemistry markedly elevates detection limits for FIX across optical and electrochemical assays. A 60‑fold LOD reduction compared to conventional ELISA, coupled with successful serum detection, positions this approach as a versatile tool for diagnosing clotting disorders and potentially other low‑concentration biomarkers.

Availability of Data and Materials

All experimental data are available without restriction.

Abbreviations

APTES

(3‑Aminopropyl)triethoxysilane

BSA

Bovine serum albumin

ELISA

Enzyme‑linked immunosorbent assay

GNP

Gold nanoparticle

HRP

Horseradish peroxidase

IDE

Interdigitated electrode

FIX

Factor IX