(T1130-01-05) CYFRA21-1 Sensing Gold Nanoparticles Based Diagnostic Kit for Non-Small Cell Lung Cancer

Purpose: Lung cancer is one of the most dangerous malignant tumours with a high rate of morbidity and mortality. The conventional methods do not offer early diagnosis, and due to poor diagnosis, there is a need to develop point-of-care testing for early and rapid diagnosis. Using nanomaterials as diagnostic agents increases diagnostic efficiency, and results are more reliable than those obtained by conventional methods like magnetic resonance imaging (MRI) and computed tomography (CT) scans. Utilizing molecular recognition components and biosensors based on nanotechnology, nano diagnostic kits present a viable method for improving the precision, speed, and cost-effectiveness of lung cancer diagnosis.

Methods: Gold nanoparticles (AuNPs) were synthesized using the citrate reduction method, resulting in a deep red colloidal solution indicative of nanoparticle formation. The synthesized AuNPs were characterized via dynamic light scattering (DLS), UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) to confirm particle size and morphology. CYFRA21-1 antibodies were conjugated to the AuNPs through chemical adsorption at an optimized pH of 9.0. Bovine serum albumin (10%w/v) was added to prevent non-specific interaction, centrifuged, and the AuNP-conjugated CYFRA21-1 pellet was resuspended in pH 7.4 R-buffer. Optimal antibody concentration for conjugation was determined through sensitivity studies using serial dilutions of CYFRA 21-1 antigen. Coating antibody concentrations on the nitrocellulose membrane were similarly optimized for enhanced sensitivity. Lateral flow immunoassay (LFIA) strips were assembled using pre-treated sample pads, conjugate pads loaded with antibody-AuNP conjugates, nitrocellulose membranes, and absorbent pads. The components were cut into 3 mm strips and housed in plastic cassettes. Testing was conducted using CYFRA 21-1 antigen across concentrations (2000 to 3.5 ng/mL), with each sample mixed in PBS buffer (pH 7.5) and applied to the strip. A visual intensity scale was used to assess test line strength, and a calibration curve was plotted to determine the sensitivity. Limit of detection (LOD) was confirmed at 3.5 ng/mL in triplicate tests. This protocol allowed for the successful development of a sensitive LFIA platform for CYFRA21-1 detection with rapid visual readout.

Results: The developed gold nanoparticles exhibited an average size of ~30 nm before conjugation and increased to ~72 nm post-conjugation, as confirmed by DLS. TEM revealed monodisperse, spherical particles with well-defined morphology. UV-visible spectroscopy showed a characteristic plasmon resonance peak at 534 nm that shifted to 541 nm post-conjugation, indicating successful antibody attachment. FTIR of conjugated nanoparticles revealed protein-associated amide bands, further confirming antibody conjugation. Conjugation optimization trials identified pH 9.0 and antibody concentration of 15,000 ng/mL as optimal for stable conjugation without aggregation, resulting in a soft pellet and stable dispersion. A higher concentration (20,000 ng/mL) caused non-specific binding and false positives, while lower concentrations (10,000 ng/mL) failed to detect low antigen levels. Nitrocellulose membrane coating concentration was optimized to 1.8 mg/mL for the CYFRA 21-1 antibody to get optimum sensitivity. The final conjugate pad was prepared using conjugated nanoparticles with a working optical density of 15, ensuring optimal flow and band visibility. The assembled LFIA strips were tested with serial dilutions of CYFRA 21-1 antigen (2000 to 3.5 ng/mL). A distinct test line was observed across all concentrations, with the most intense band at 2000 ng/mL and faint but visible bands down to 3.5 ng/mL. Testing with CYFRA 21-1 negative serum samples showed no false positives, demonstrating high assay specificity. A sensitivity curve was plotted based on visual band intensities, which correlated with antigen concentration. These results confirm the successful development of a specific and sensitive LFIA platform capable of detecting CYFRA 21-1 biomarker at clinically relevant levels (LOD: 3.5 ng/mL), offering potential for early detection of lung cancer biomarkers in point-of-care settings.

Conclusion: The developed nano-diagnostic LFIA kit utilizing gold nanoparticles conjugated with CYFRA21-1 antibodies demonstrated effective, dose-dependent colorimetric detection of lung cancer biomarker, enabling rapid, affordable, and semi-quantitative screening. Its potential for early diagnosis in low-resource settings is promising. Future improvements include validation with clinical samples, integration of multiple biomarkers to enhance sensitivity and specificity, and use of digital tools for quantification. Adapting the assay for non-invasive samples and optimizing signal amplification strategies could further improve utility. For clinical translation, long-term stability, reproducibility, and scalable production must be addressed, advancing its application as a reliable point-of-care diagnostic tool.

References: McFarland AD, Haynes CL, Mirkin CA, Van Duyne RP, Godwin HA. Color My Nanoworld. J Chem Educ. 2004 Apr 1;81(4):544A

Acknowledgements: Government of India, for providing GAT-B exam fellowship to D.N. Paramcare Life Sciences, Vapi, Gujarat, for sharing valuable inputs to D.N. during internship.

Funding: Department of Biotechnology, Ministry of Science and Technology, Government of India. Conflict of Interest Statements: All authors declare no conflict of interest.

Figure 1. Testing with antigen (positive samples)

Figure 2. Testing with negative serum samples