The detection of tumor markers in body fluids is crucial for the screening, diagnosis, and prognosis analysis of cancer. Hence, the sensitivity of tumor biomarker screening is highly demanded in detection. Currently, several detection techniques are available, such as a fluorescence analysis, surface-enhanced Raman scattering, electrochemical luminescence, and an electrochemical analysis. However, these methods have certain limitations, such as low sensitivity, poor stability, complex processes, and long reaction time. In recent years, the imaging technique combined with precious metal and dark-field microscopy has gained popularity in the field of highly sensitive biochemical detection due to its high spatiotemporal resolution and independence of signal reporter molecules. Gold nanorods (AuNRs) are anisotropic nanomaterials that show two types of plasmon resonance—longitudinal plasmon resonance and transverse plasmon resonance—in which the longitudinal LSPR plays a dominant role in the detection, while the transverse LSPR mode is always neglected. Herein, polarized light, which is perpendicular to the AuNRs, is designed to stimulate the transverse plasma resonance of the AuNRs to detect biomarkers in a microfluidic chip. In this work, Vascular Endothelial Growth Factor (VEGF₁₆₅) is used as the testing biomarker to demonstrate the feasibility of this method. With the presence of VEGF₁₆₅ in the sample solution, AuNRs will capture the gold nanoparticles due to the antibody–antigen–antibody switched structure, inducing the change in the polarized plasma resonance property. This method achieves a detection limit of 10 pg/ml for VEGF₁₆₅, which is lower than most of the reported methods. The results show that the method based on the combination of a microfluidic chip and dark-field microscopic image has excellent sensitivity and has significant potential in an early cancer diagnosis and prognosis analysis.

Abstract:

Chemosensors based on Schiff bases are pivotal in environmental and biological applications, serving to identify specific metal ions at trace levels. Despite the distinctive importance of thiophene-based molecules in medicinal contexts, the number of reported chemosensors utilizing these moieties remains limited.

In this study, we present the synthesis and characterization of a novel Schiff base sensor (TBH), derived from thiophene-2-carboxaldehyde and benzil. We investigate its application as a selective relay probe for the detection of Zn²⁺ and Fe²⁺ ions.

The introduction of Zn²⁺ to TBH resulted in a significant enhancement in fluorescent intensity, attributed to the formation of a 1:1 TBH–Zn²⁺ complex, with no response observed for other cations, including Mg²⁺, Ba²⁺, Cd²⁺, Cu²⁺, Co²⁺, Mn²⁺, Cr³⁺, Hg²⁺, Sn²⁺, La³⁺, Ca²⁺, Na⁺, K⁺, and particularly Fe²⁺. Furthermore, Fe²⁺ induced fluorescence quenching in the TBH–Zn²⁺ system, forming a 1:1 MY–Fe²⁺ complex. The TBH-Zn²⁺ complex demonstrates potential as a secondary sensor for Fe²⁺ ions. The sensor's signal change is based on the chelation-enhanced fluorescence (CHEF) effect of TBH–Zn²⁺, coupled with the inhibition of photoinduced electron transfer (PET).

Moreover, the rapid and selective features of the proposed sensor make it promising for the precise monitoring of Zn²⁺and Fe²⁺ in biological and environmental research.

AI biosensors are devices that can detect and measure biological or chemical signals of interest, such as glucose, DNA, hormones, toxins, pathogens, etc. They have many applications in various fields, such as healthcare, environmental monitoring, food safety, biodefense, and bioengineering. However, AI biosensors also pose some regulatory and ethical challenges that need to be addressed before they can be widely used and accepted by society. Some of these challenges are safety and reliability, privacy and data protection, social and cultural implications, innovation, and regulation. AI biosensors are constantly evolving and innovating with new technologies, materials, methods, or applications. This may pose challenges for the existing regulatory frameworks and authorities that may not be able to keep up with the pace and scope of innovation. AI biosensors should balance between innovation and regulation, and we should ensure that they are developed and used in a responsible and sustainable manner. Various stakeholders, such as researchers, regulators, policy makers, industry partners, civil society groups, and end-users should engage with AI biosensors to foster dialogue, collaboration, and public trust. Proposed in April 2021 and expected to enter into force in 2025, the European Union Artificial Intelligence Act (EU AI Act) will be the first EU regulatory framework for AI and could serve as a law model for the regulation of AI biosensors. There are some scattered international instruments and frameworks that address some of the ethical, legal, and social issues related to biosensors. States and the World Health Organization (WHO), with its constitutional mandate to deal with global public health, should regulate the use of AI-biosensors and adopt legally binding rules and international standards in this sensitive field.

Introduction

Incorporating SERS tags into clinical diagnostics holds the potential to increase the sensitivity of diagnostic techniques. Gold petal-like gap-enhanced Raman tags (pGERTs) exhibit an exceptionally high Raman signal. However, the complexity of pGERT functionalization with recognition molecules limits their application in immunoassays. In this study, we have developed a novel method for biofunctionalization of pGERTs with monoclonal antibodies specific to the SARS-CoV-2 spike protein, employing controlled assembly of polydopamine (PDA) on the nanoparticle surface.

Methods

CTAC-coated pGERTs, containing 4-nitrobenzenethiol (4-NBT), were synthesized and characterized. Prior to PDA layer deposition, pGERTs were pre-functionalized with various polymers. Conjugation of PDA-coated pGERTs with monoclonal antibodies specific for S-protein was conducted, and the success of conjugation was confirmed via a dot-immunoassay.

Results

It was demonstrated that replacing CTAC with polystyrene sulfonate on the surface of 60 nm pGERTs allows for controlled PDA growth, resulting in a 3-4 nm-thick PDA layer. Spectrophotometry revealed a broadening of the absorption spectrum and a 4 nm red-shift in the absorption peak of PDA-coated pGERTs. The SERS spectrum of these particles exhibited all characteristic bands of 4-NBT. A dot-immunoassay confirmed the selective staining of the S-protein at varying concentrations (1, 10, 50 μg/mL) by the resulting conjugates.

Conclusions

We have developed a method for the biofunctionalization of pGERTs with monoclonal antibodies specific to the S-protein. The obtained results offer potential applications in the development of SERS-based immunoassay systems.

Acknowledgements

This research was supported by the Russian Science Foundation, grant number № 23-24-00246 (https://rscf.ru/project/23-24-00246/).

Introduction

Accurately detecting bacterial endotoxin levels is essential for effective pharmaceutical quality control. Endotoxins refers to lipopolysaccharides that are present in the outer membrane of Gram-negative bacteria. They elicit a robust inflammatory response upon exposure. Gold nanoclusters (AuNCs) show potential as fluorescent nanotags due to their ease of synthesis, wide Stokes shift, and exceptional photostability. This study introduces fluorescent AuNCs for endotoxin detection for the first time.

Methods

Glutathione-stabilized gold nanoclusters (GSH-AuNCs) were synthesized and characterized. Lipopolysaccharide extraction from Pseudomonas putida bacterial biofilms was carried out. The relationship between the change in fluorescence intensity of GSH-AuNCs and the concentration of P. putida endotoxin was established. The micelle characterization of the endotoxin was conducted using dynamic light scattering (DLS).

Results

Orange-emitting 1.5 nm GSH-AuNCs were produced. Upon interacting with endotoxin micelles, the fluorescence signal increased and the emission peak shifted by 7-10 nm towards the blue end of the spectrum. This was attributed to the phenomenon of aggregation-induced emission (AIE). The detection limit for the endotoxin in aqueous solutions was determined to be 400 ng/ml. The size of the endotoxin micelles was measured to be 62 ± 15 nm.

Conclusions

We have developed a nanosensor based on fluorescent AuNCs for endotoxin detection without requiring extra recognition molecules. The detection principle relies on the modulation of the fluorescence properties of GSH-AuNCs in the presence of endotoxin micelles. This nanosensor holds potential for endotoxin detection in pharmaceutical preparations and buffer solutions.

Acknowledgements

This research was supported by the Russian Science Foundation, grant number № 23-24-00246 (https://rscf.ru/project/23-24-00246/).