A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

A dual-binding magnetic immunoassay to predict spontaneous preterm birth.

Complications posed by preterm birth (delivery before 37 weeks of pregnancy) are a leading cause of newborn morbidity and mortality. The previous discovery and validation of an algorithm that includes maternal serum protein biomarkers, sex hormone-binding globulin (SHBG), and insulin-like growth factor-binding protein 4 (IBP4), with clinical factors to predict preterm birth represents an opportunity for the development of a widely accessible point-of-care assay to guide clinical management. Toward this end, we developed SHBG and IBP4 quantification assays for maternal serum using giant magnetoresistive (GMR) sensors and a self-normalizing dual-binding magnetic immunoassay. The assays have a picomolar limit of detections (LOD) with a relatively broad dynamic range that covers the physiological level of the analytes as they change throughout gestation. Measurement of serum from pregnant donors using the GMR assays was highly concordant with those obtained using a clinical mass spectrometry (MS)-based assay for the same protein markers. The MS assay requires capitally intense equipment and highly trained operators with a few days turnaround time, whereas the GMR assays can be performed in minutes on small, inexpensive instruments with minimal personnel training and microfluidic automation. The potential for high sensitivity, accuracy, and speed of the GMR assays, along with low equipment and personnel requirements, make them good candidates for developing point-of-care tests. Rapid turnaround risk assessment for preterm birth would enable patient testing and counseling at the same clinic visit, thereby increasing the timeliness of recommended interventions.

Cortisol Detection in Undiluted Human Serum Using a Sensitive Electrochemical Structure-Switching Aptamer over an Antifouling Nanocomposite Layer.

There is a strong and growing need to monitor stress biomarkers in vivo for real-time emotional and wellness assessment. Toward this, we report a reagent-free electrochemical aptasensor with a nanocomposite antifouling layer for sensitive and continuous detection of cortisol in human serum. A thiolated, methylene blue (MB)-tagged conformation-switching aptamer was immobilized over a gold nanowire (AuNW) nanocomposite to capture cortisol and generate a signal proportional to the cortisol concentration. The signal is recorded through differential pulse voltammetry (DPV) and chronoamperometry. The aptasensor exhibited a sensitive response with 0.51 and 0.68 nM detection limits in spiked buffer and undiluted serum samples, respectively. Interference from other structurally similar analogs, namely, epinephrine and cholic acid, was negligible (<10%). The developed nanocomposite-based aptasensor showed excellent stability in undiluted human serum, outperforming several other nanocomposite materials even after prolonged exposure. This work lays the foundation for new biosensor formats such as implantable and wearable sensors.

Giant magnetoresistive biosensors for real-time quantitative detection of protease activity.

Proteases are enzymes that cleave proteins and are crucial to physiological processes such as digestion, blood clotting, and wound healing. Unregulated protease activity is a biomarker of several human diseases. Synthetic peptides that are selectively hydrolyzed by a protease of interest can be used as reporter substrates of unregulated protease activity. We developed an activity-based protease sensor by immobilizing magnetic nanoparticles (MNPs) to the surface of a giant magnetoresistive spin-valve (GMR SV) sensor using peptides. Cleavage of these peptides by a protease releases the magnetic nanoparticles resulting in a time-dependent change in the local magnetic field. Using this approach, we detected a significant release of MNPs after 3.5 minutes incubation using just 4 nM of the cysteine protease, papain. In addition, we show that proteases in healthy human urine do not release the MNPs, however addition of 20 nM of papain to the urine samples resulted in a time-dependent change in magnetoresistance. This study lays the foundation for using GMR SV sensors as a platform for real-time, quantitative detection of protease activity in biological fluids.

A CMOS Magnetoresistive Sensor Front-End With Mismatch-Tolerance and Sub-ppm Sensitivity for Magnetic Immunoassays.

Magnetic biosensing is an emerging technique for ultra-sensitive point-of-care (PoC) biomolecular detection. However, the large baseline-to-signal ratio and sensor-to-sensor mismatch in magnetoresistive (MR) biosensors severely complicates the design of the analog front-end (AFE) due to the high dynamic range (DR) required. The proposed AFE addresses these issues through new architectural and circuit level techniques including fast settling duty-cycle resistors (DCRs) to reduce readout time and a high frequency interference rejection (HFIR) sampling technique embedded in the ADC to relax the DR requirement. The AFE achieves an input-referred noise of 46.4 nT/√Hz, an input-referred baseline of less than 0.235 mT, and a readout time of 11 ms while consuming just 1.39 mW. Implemented in a 0.18 µm CMOS process, this work has state-of-the-art performance with 22.7× faster readout time, >7.8× lower baseline, and 2.3× lower power than previously reported MR sensor AFEs.