NLRP3, IL-1β, and Caspase-1 Gene Transcript Identification and Expression by QCM-D in Obese Children

Abstract

Background. Obesity in children is highly prevalent in Mexican population. Adipose tissue has been related to specific pro- and anti-inflammatory cytokine and inflammasome gene and protein expression patterns. Actually, there is no existing biosensor for detecting gene expression patterns in children with obesity. The quartz crystal microbalance with dissipation monitoring (QCM-D) has been used as a transducer for DNA biosensor design. Results. In this study, the gene expression pattern of IL-1β, NLRP3, and CASPASE-1 in children with obesity was successfully determined by means of QCM-D. Gene expression patterns were validated with those obtained by quantitative polymerase chain reaction (qPCR), a validated molecular biology technique for gene expression quantification. QCM-D analysis of the detected mass corresponding results for each of the genes showed a major detected mass for IL-1β, followed by similar NLRP3 and constitutive gene 18S deposited mass and a smaller deposited mass for CASPASE-1. Surprisingly, when comparing mRNA gene expression results for NLRP3, IL-1β, and CASPASE-1 obtained with qPCR and QCM-D, similar patterns were found, revealing greatest expression of IL-1β, followed by NLRP3, with CASPASE-1 being the molecule of least expression in the group of children with obesity. AFM images illustrate the step-by-step changes that took place on the quartz surface. Conclusions. QCM-D proved successfully for determining the gene transcripts and expression of NLRP3, IL-1β, and CASPASE-1 in children with obesity, with similar results validated by qPCR. “QCM-D decreases detection costs compared with a validated molecular biology technique.” The QCM-D biosensor developed by our group was successful for gene expression determination; in the future, it can be used for molecular diagnosis.

Biosensor of inflammation biomarkers based on electrical bioimpedance analysis on immobilized DNA without chemical modification

“…The positively charged carbon atom in carbodimides rapidly reacts with the partially negatively charged oxygen in the 5′-phosphate group forming an active phosphodiester. This chemistry was previously reported by our group [ 19 ]. Diagram of functionalized gold surface quartz crystal and representation of the cDNA anchorage and as hybridization is expected is shown in Fig 2 .…”

“…It should be noted that unlike Marquette's and Mousavisani's groups that worked with synthetic DNA hybridization, our results show different impedance when hybridizing specific oligonucleotides to identify the expression of genes related to inflammation in a population of obese Mexican children [ 19 ]. In a study that could be considered as a previous reference, Ma's group reported a correlation between impedance and DNA fragment length, proposing the use of an electrical system to validate and characterize PCR products [ 25 ].…”

“…EBiS is recognized as a safe, fast, reusable, easy, inexpensive and simple to perform technique, requiring no detection tags, also allowing for miniaturization [ 14 , 20 ]. Due to these advantages, EBiS is the technique preferred for label-free detections and for bio-recognitions events such as protein immobilization, antibody affinity, enzyme activity, DNA hybridization detection, and to monitor DNA damage [ 7 , 11 , 19 , 21 ].…”

“…In this study, we demonstrate that three genes involved in the inflammation process can be measured by means of EBiS on immobilized cDNA. The immobilization of cDNA in a functionalized gold surface has been successfully evaluated by our research group through Atomic Force Microspocpy (AFM) characterization; the surface chemistry was achieved using human DNA as probe with no chemical modification [ 19 ].…”

Strategies for the Accurate Measurement of the Resonance Frequency in QCM-D Systems via Low-Cost Digital Techniques

“…Focusing on the direct resonance frequency measurement, a more versatile widespread technique is the Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]; this technique is based on the simultaneous measurement of the sensor resonance frequency and of its dissipation factor (i.e., the reciprocal of the quartz quality factor) during a transient response triggered by short electric excitations.…”