Early diagnosis and monitoring are essential for the effective treatment and survival of patients with different types of malignancy. To this end, the accurate and sensitive determination of substances in human biological fluids related to cancer diagnosis and/or prognosis, i.e., cancer biomarkers, is of ultimate importance. Advancements in the field of immunodetection and nanomaterials have enabled the application of new transduction approaches for the sensitive detection of single or multiple cancer biomarkers in biological fluids. Immunosensors based on surface-enhanced Raman spectroscopy (SERS) are examples where the special properties of nanostructured materials and immunoreagents are combined to develop analytical tools that hold promise for point-of-care applications. In this frame, the subject of this review article is to present the advancements made so far regarding the immunochemical determination of cancer biomarkers by SERS. Thus, after a short introduction about the principles of both immunoassays and SERS, an extended presentation of up-to-date works regarding both single and multi-analyte determination of cancer biomarkers is presented. Finally, future perspectives on the field of SERS immunosensors for cancer markers detection are briefly discussed.

OoC systems employing human cells mirror the functionality of human organs and faithfully simulate their physiological microfluidic environment. Despite the potential of OoC technology in emulating tissue complexity, a significant gap persists in the continuous real-time monitoring of cellular behaviors and their responses to external stimuli, arising from the lack of biosensors integrated onto OoC microfluidic platforms. Addressing this limitation constitutes the primary objective of this study. By developing and incorporating biosensors onto a modular integrated OoC platform, we aim to enable the monitoring of changes taking place in the cellular environment under various stimuli in real time. An in-series modular integration of a biosensor array into an OoC platform is demonstrated herein, along with its potential to sustain human cell proliferation and accommodate the detection of IL-6, as an example of a mediator protein secreted as part of the immune response to inflammation. The implementation of commercially fabricated PCB components also addresses the issue of cost efficiency and manufacturing scaling-up of sensor-integrated OoCs. This advancement will not only enhance the accuracy and reliability of preclinical studies, but also pave the way for improved drug development and disease treatment.

Glutathione and malondialdehyde are two compounds commonly used to evaluate the oxidative stress status of an organism. Although their determination is usually performed in blood serum, saliva is gaining ground as the biological fluid of choice for oxidative stress determination at the point of need. For this purpose, surface-enhanced Raman spectroscopy (SERS), which is a highly sensitive method for the detection of biomolecules, could offer additional advantages regarding the analysis of biological fluids at the point of need. In this work, silicon nanowires decorated with silver nanoparticles made by metal-assisted chemical etching were evaluated as substrates for the SERS determination of glutathione and malondialdehyde in water and saliva. In particular, glutathione was determined by monitoring the reduction in the Raman signal obtained from substrates modified with crystal violet upon incubation with aqueous glutathione solutions. On the other hand, malondialdehyde was detected after a reaction with thiobarbituric acid to produce a derivative with a strong Raman signal. The detection limits achieved after optimization of several assay parameters were 50 and 3.2 nM for aqueous solutions of glutathione and malondialdehyde, respectively. In artificial saliva, however, the detection limits were 2.0 and 0.32 μM for glutathione and malondialdehyde, respectively, which are, nonetheless, adequate for the determination of these two markers in saliva.

Oxygen plasma micro-nanostructured poly(methyl methacrylate) (PMMA) slides were modified through silver microparticle deposition to create microarray substrates that enhance the emitted fluorescence intensity. Silver deposition relied on a commercially available reagent and was completed in two 30-min incubation cycles of the substrate with the reagent. The fluorescence enhancement achieved using these substrates over flat PMMA slides was determined through the development of a microarray for the multiplexed detection of four mycotoxins, aflatoxin B1, ochratoxin A, fumonisin B1, and deoxynivalenol. It was shown that the implementation of silver-plated oxygen plasma micro-nanotextured PMMA substrates increased the signals obtained for aflatoxin B1 and ochratoxin A by approximately 2.8 times, 5.6 times for deoxynivalenol, and 16-times for fumonisin B1, compared to flat PMMA substrates. Most notably, this signal increase was not accompanied by a significant increase in the non-specific signal. In addition, the spot repeatability both across a single slide as well as between different slides was high, with coefficients of variation lower than 12%. The slides were also stable for at least three months, thus offering a microarray substrate with improved properties compared to standard glass slides, regarding both the absolute spot fluorescence intensity and between spots repeatability.

Nanostructured noble metal surfaces enhance the photoluminescence emitted by fluorescent molecules, permitting the development of highly sensitive fluorescence immunoassays. To this end, surfaces with silicon nanowires decorated with silver nanoparticles in the form of dendrites or aggregates were evaluated as substrates for the immunochemical detection of two ovarian cancer indicators, carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4). The substrates were prepared by metal-enhanced chemical etching of silicon wafers to create, in one step, silicon nanowires and silver nanoparticles on top of them. For both analytes, non-competitive immunoassays were developed using pairs of highly specific monoclonal antibodies, one for analyte capture on the substrate and the other for detection. In order to facilitate the identification of the immunocomplexes through a reaction with streptavidin labeled with Rhodamine Red-X, the detection antibodies were biotinylated. An in-house-developed optical set-up was used for photoluminescence signal measurements after assay completion. The detection limits achieved were 2.5 U/mL and 3.12 pM for CA125 and HE4, respectively, with linear dynamic ranges extending up to 500 U/mL for CA125 and up to 500 pM for HE4, covering the concentration ranges of both healthy and ovarian cancer patients. Thus, the proposed method could be implemented for the early diagnosis and/or prognosis and monitoring of ovarian cancer.

Oxidative stress refers to the overproduction of reactive oxygen species and is often associated with numerous pathological conditions. Superoxide dismutase (SOD) is a widely used enzyme for evaluating oxidative stress, with numerous methods being developed for its detection in biological specimens like blood, urine, and saliva. In this study, a simple metal-assisted chemical etching method was employed for the fabrication of nanostructured silicon surfaces decorated with either silver dendrites or silver aggregates. Those surfaces were used as substrates for the immunochemical determination of SOD in synthetic saliva through surface-enhanced Raman spectroscopy (SERS) and surface-enhanced fluorescence (SEF). The immunoassay was based on a 3-step competitive assay format, which included, after the immunoreaction with the specific anti-SOD antibody, a reaction with a biotinylated secondary antibody and streptavidin. Streptavidin labeled with peroxidase was used in combination with a precipitating tetramethylbenzidine substrate for detection through SERS, whereas for SEF measurements, streptavidin labeled with the fluorescent dye Rhodamine Red-X was utilized. Both immunoassays were sensitive, with a detection limit of 0.01 μg/mL and a linear dynamic range from 0.03 to 3.3 μg/mL, enabling the evaluation of the oxidative stress status of an organism.