Cesar Sanchez-Huertas

Multiplexed optical sensing using integrated photonic platforms demands scalable interrogation architectures that do not compound hardware complexity with increasing numbers of sensing channels. Here, we demonstrate simultaneous readout of two cascaded silicon nitride (Si₃N₄) racetrack microring resonators (MRR) with different free spectral ranges (FSR), using a single electro-optic optical frequency comb (EO-OFC) generated from one continuous-wave laser source. By tuning the radio-frequency (RF) modulation frequency to match the spectral separation between selected resonances of the two rings, individual comb lines are aligned to each resonator, enabling independent and simultaneous resonance tracking without wide-range wavelength scanning. A programmable waveshaper demultiplexes the carrier and first-order sideband channels to dedicated photodetectors, allowing real-time monitoring of both rings in parallel. The system was validated by bulk refractive-index sensing with glycerol solutions at five concentrations (0.039–0.6% v/v). Both rings achieved sensitivities in the range of 26.8–38.5 nm/RIU and limits of detection (LoD) on the order of 10-5 RIU (2.15–2.96 × 10-5 RIU), with OFC-multiplexed performance matching single-wavelength baseline measurements on the same chip. The carrier and sideband channel assignments were interchangeable, confirming architectural flexibility. These results establish EO-OFC interrogation as a viable, scalable route to multiplexed microring resonator sensing, with direct implications for parallel biosensing, environmental monitoring, and food safety applications.

Accurate diagnosis of myocardial injury is crucial in clinical practice, as it enables prompt treatment, reduces the risk of complications, and improves patient outcomes. Examining cardiac troponin composition offers distinct advantages as it provides valuable insights that help differentiate between ischaemic and non-ischaemic causes of cardiac injury, thereby directly enhancing clinical assay specificity. Current high-sensitivity troponin assays target all circulating forms of either troponin I or T but are unable to distinguish individual forms or specific combinations. This is an issue as troponin is also elevated in non-cardiac conditions, and emerging evidence suggests that the ratio between troponin I and TIC complex isoforms may serve as an indicator for differentiating acute from chronic myocardial injury. Here, we introduce a methodology utilising a label-free Surface Plasmon Resonance biosensor to detect and differentiate between troponin I and complex isoforms. Unlike traditional immunoassay, which requires indirect labelling with secondary antibodies to detect these biomarkers, this method provides a real-time detection of these isoforms, as each antibody interaction with the specific analytes provides a quantitative reading. We demonstrated that this method can detect complexed and free troponin I in varying ratios. This method has the potential to be developed into point-of-care testing and opens avenues for isoform-specific prognostic assays.

Silicon photonic microring resonators have emerged as promising sensors for Point‐of‐Care applications, where the readout of one or many biomarkers at once is required. In the context of rapid heart attack detection, a limit of detection reaching an ultra‐low concentration of biomarkers is needed, however, such sensors are prone to fundamental noise influence in optical systems which can potentially jeopardize sensor readings. While noise reduction has previously been explored with the Pound–Drever–Hall (PDH) technique, its full implementation in microring biosensors has not been realized due to the complexity of the setup. Recent innovations in photonic integration and compatibility with MEMS structures have sparked new interest in validating PDH's potential to be used with chip‐scale sensors. By enabling the signal readout of microrings through their phase response, instead of power transmission, the impact of optical noise can be greatly reduced. This study explores a proof‐of‐concept for this system against the cardiac troponin biomarker, demonstrating the sensor's capacity for selective measurement down to a limit of 10 ng mL⁻¹ while using frequency locking. An improved limit of detection for this system is achieved, down to 5.03 × 10⁻⁷ RIU, which is two orders of magnitude improved compared to the equivalent sensing based on intensity alone.

Ovarian cancer (OC) is one of the most common gynecological cancers

The bimodal waveguide (BiMW) biosensor is an innovative common path interferometric sensor based on the evanescent field detection principle. This biosensor allows for the direct detection of virtually any biomolecular interaction in a label-free scheme by using specific biorecognition elements. Due to its inherent ultrasensitivity, it has been employed for the monitoring of relevant nucleic-acid sequences such as mRNA transcripts or microRNAs present at the attomolar–femtomolar concentration level in human samples. The application of the BiMW biosensor to detect these nucleic acids can be a powerful analytical tool for diagnosis and prognosis of complex illnesses, such as cancer, where these biomarkers play a major role. The BiMW sensor is fabricated using standard silicon-based microelectronics technology, which allows its miniaturization and cost-effective production, meeting the requirements of portability and disposability for the development of point-of-care (PoC) sensing platforms. In this chapter, we describe the working principle of the BiMW biosensor as well as its application for the analysis of nucleic acids. Concretely, we show a detailed description of DNA functionalization procedures and the complete analysis of two different RNA biomarkers for cancer diagnosis: (1) the analysis of mRNA transcripts generated by alternative splicing of Fas gene, and (2) the detection of miRNA 181a from urine liquid biopsies, for the early diagnosis of bladder cancer. The biosensing detection is performed by a direct assay in real time, by monitoring the changes in the intensity pattern of the light propagating through the BiMW biosensor, due to the hybridization of the target with the specific DNA probe previously functionalized on the BiMW sensor surface.

Label-free plasmonic biosensors have demonstrated promising capabilities as analytical tools for the detection of virtually any type of biomarker. They are presented as good candidates for precision diagnostics since they offer highly sensitive, cost-effective solutions that can be used in any clinical or laboratory setting without the need for specialized trainees. How-ever, different surface functionalization protocols are required depending on the nature of the biorecognition element, limit-ing their capabilities for integrated multibiomarker detection. Here, we present a simple, yet efficient one-step immobiliza-tion approach that is common for both DNA probes and antibodies. Our immobilization approach relies on the incorpora-tion of polyA blocks in both nucleic acid probes and antibodies, which specifically interact with the gold surface with suffi-cient strength to generate dense and highly ordered monolayers. We have demonstrated excellent performance of our uni-versal functionalization method, showing limits of detection and quantification in the pM-nM range. Moreover, it was able to reduce up to 50% the background signal from undiluted serum samples compared to conventional methods, demonstrat-ing the immense potential of this strategy for the direct analysis of human biofluids, essential for rapid point-of-care diag-nostics. The polyA-based immobilization approach is a promising alternative for the biofunctionalization of microarray-format sensors and their application to multiplexed diagnostic assays.

Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most severe infections encountered in immunodepressed individuals. The diagnosis of Pneumocystis pneumonia continues to be challenging due to the absence of specific symptoms in infected patients. Moreover, the standard diagnostic method employed for its diagnosis involves mainly PCR-based techniques, which besides being highly specific and sensitive, require specialized personnel and equipment and are time-consuming. Our aim is to demonstrate an optical biosensor methodology based on surface plasmon resonance to perform such diagnostics in an efficient and decentralized scheme. The biosensor methodology employs poly-purine reverse-Hoogsteen hairpin probes for the detection of the mitochondrial large subunit ribosomal RNA (mtLSU rRNA) gene, related to P. jirovecii detection. The biosensor device performs a real-time and label-free identification of the mtLSU rRNA gene with excellent selectivity and reproducibility, achieving limits of detection of around 2.11 nM. A preliminary evaluation of clinical samples showed rapid, label-free and specific identification of P. jirovecii in human lung fluids such as bronchoalveolar lavages or nasopharyngeal aspirates. These results offer a door for the future deployment of a sensitive diagnostic tool for fast, direct and selective detection of Pneumocystis pneumonia disease.

Alternative Splicing of messenger RNA precursors is a key process in gene regulation, contributing to the diversity of proteomes by the alternative selection of exonic sequences. Most cancers are associated with alterations in this mechanism, enhancing their proliferation and survival, and can be employed as cancer biomarkers. Label-free optical biosensors are ideal tools for the highly sensitive and label-free analysis of nucleic acids. However, their application for alternative splicing analysis has been hampered due to the formation of complex and intricate long-range base-pairing interactions which make the direct detection in mRNA isoforms difficult. To solve this bottleneck, we introduce a methodology for the generation of length-controlled RNA fragments from purified total RNA, which can be easily detected by the biosensor. The methodology seizes RNase H enzyme activity to degrade the upstream and downstream RNA segments flanking the target sequence upon hybridization to specific DNA-oligos. It allows the fast and direct monitoring of Fas gene alternative splicing in real-time employing a Surface Plasmon Resonance (SPR) biosensor. We demonstrate the selective and specific detection of mRNA fragments in the pM-nM concentration range, reducing quantification errors and showing 81% accuracy when compared to RT-qPCR. The site-specific cleavage outperformed a random RNA hydrolysis by increasing the detection accuracy in 20%, making this methodology particularly appropriate for label-free quantification of alternative splicing events in complex samples.