NEW YORK (GenomeWeb) – Researchers from the University of Michigan have published early data describing their adaptation of a technology called localized surface plasmon resonance (LSPR)-based biosensing, into a method for rapid, real-time, multiplex monitoring of serum cytokines.
In the study, published last month in the journal ACS Nano, the group also demonstrated that the method was able to monitor the inflammatory response of two infants after cardiopulmonary bypass surgery, a proof of concept of its future clinical potential.
Pengyu Chen, the study's first author, told GenomeWeb this week that he has been working with LSPR through his graduate studies, with the idea that its ability to detect analyte binding in real time could open up new clinical applications for immunoassays that current technologies do not allow, for example, the precise monitoring of immune status during the course of a disease as manifest in changing levels of cytokines.
With LSPR, the surface binding of a target analyte (or analytes) is detected via a shift in the photon absorbing and scattering behavior of oscillating conduction-band electrons localized on the surfaces of metallic nanoparticles.
This allows real-time refractometric detection of molecular binding, and potentially more rapid and less sample-consuming analysis than with fluorescence-based immunoassays.
While the technique is not new, and has drawn attention widely as a potential alternative to fluorescence-based immunoassay methods, implementing it clinically as a biomarker detection and quantification tool is still in its infancy, according to the study authors.
In their study, the Michigan group set out to fabricate and test an LSPR microarray device that would allow for high-throughput detection of multiple cytokine biomarkers in very small serum samples.
The team's resulting LSPR chip consists of eight parallel microfluidic channels running across six stripes of antibody-functionalized gold nanorod ensembles that they showed could quantitate cytokines down to 5-20 pg/mL in a 1uL serum sample.
By changing the microfluidic patterning conditions used to plot these nanorod stripes out, the size and number of the microarrays can be tuned much higher, the authors wrote, meaning the technology has the potential for much higher multiplexing without being limited by antibody cross reactions. However, for their study, the team conjugated the array's gold nanorod stripes with antibodies against just six different cytokines — IL-2, IL-4, IL-6, IL-10, IFN-y, and TNF-a.
After some calibration experiments, the researchers performed an initial validation of the six-cytokine chip using a set of serum samples spiked with different mixtures of these six targets.
Translating the LSPR array readout into a measure of the concentration of target analytes, the researchers reported that they found no statistically significant difference between the measured cytokine concentrations and the values expected based on the spiking concentrations.
Next, the group moved on to a second validation step, comparing the LSPR array method with the existing gold standard — ELISA. The researchers prepared healthy donor serum samples spiked with a mixture of the six target cytokines at a range of concentrations covering the full dynamic range of the LSPR assay. They then compared the results of multiplex detection using the LSPR method to results from parallel singleplex ELISA-based testing of the same spiked serum samples.
According to the group, there was an "excellent" linear correlation — a coefficient of determination of 0.97 — between results generated from the LSPR method and the ELISAs.
Finally, the researchers sought to demonstrate, at least initially, the clinical utility of the approach in the context of monitoring inflammatory response, in this case in neonates after cardiothoracic surgery.
The group collected serum samples from two babies at several time points starting before surgery and up to four days afterward, and used the six-target LSPR microarray immunoassay to quantify circulating cytokines levels in all the samples.
In both infants, the team saw increased levels of both IL-6 and Il-10 on day one after surgery, along with other elevations on day one and two. All the elevated cytokines then showed a return to preoperative levels on days three and four, which matched what has been seen in previous reports.
The process of performing the assays, from loading, incubation, and washing of samples through a 10-fold replicated detection for each sample using the entire biosensor array, took only 40 minutes.
Moving forward, Chen said that the LSPR array is now being used in an expanded trial at the University of Michigan to investigate and validate a panel of immune markers for pediatric sepsis prediction and monitoring.
"Currently we are trying to stratify maybe 30 or more patient samples, using the LSPR platform to measure the concentration of a set of biomarkers to see which patients have high [versus] low risk of developing sepsis," he said.
"Then in those [babies] identified to have high risk we will further measure cytokines to track the immune status," to try to inform more personalized treatment, he added.