Specificity validation of EC sensor
The EC sensors are 16 integrated gold electrode arrays. Probes for mRNA and protein are pre-coated onto different electrodes. Sandwich assay are carried out for both salivary mRNA and protein by spiking the standard in vitro translated (IVT) RNA and protein standard into saliva ().
Figure 1 Electrochemical (EC) sensor for multiple salivary biomarker detection. (a) Illustration for array of electrodes with both mRNA (left) and protein (right) detection. (b) Amperometric detection of IL-8 mRNA probe with (1) 5 nM IL-8 IVT RNA, (2) 5 nM S100A8 (more ...)
The specificity experiments are carried out. For IL-8 mRNA probe, the comparison is between complementary IL-8 mRNA and a salivary internal reference gene S100A8 RNA. For IL-8 protein probe, the comparison is between IL-8 protein and another oral cancer marker IL-1β protein. Both the raw data of amperometric detection () and the bar charts () are shown. With the IL-8 mRNA hairpin probe(18
), 5 nM IL-8 mRNA generates −904 nA current (mean value). 5 nM S100A8 only results in −103 nA current (mean value), which is very close to the blank control (−98 nA). For protein assay, the current level for 12.5 ng/ml complementary IL-8 protein is −298 nA, while for IL-1β protein it is only −50 nA (blank control is −33 nA). These results indicate the good specificity of the EC sensors.
Application of cyclic square wave electrical field (csw E-field) during detection
Multiplexing assay of mRNA and protein simultaneously has the advantage of high accuracy(10
). However, the traditional PCR and ELISA technologies have difficulties in obtain both sensitivity and specificity under the same condition. Instead of applying different buffer and temperature to optimize the assay, cyclic square wave electrical field (csw E-field) provides more effective and versatile way to control the assay. With the csw E-field, both the hybridization and protein binding are finished on the same chip within minutes, while previously these processes have to be done separately and the incubation time varies from 1-24 hours. Meanwhile, the negative potential in csw E-field removes the weak non-specific binding which generate high specificity. csw E-field also results in good mixing during the incubation, which accelerates the binding process as well.
The results of csw E-field with both IL-8 mRNA and IL-8 protein are shown in . From the studies in total reaction time, there exists an optimized condition for the csw E-field by spiking the standard in vitro translated (IVT) RNA and protein standard into saliva. The signal-to-background ratio (SBR) increases at the very beginning and reach to the highest SBR of about 10. Long time of incubation does not contribute to further improvement in SBR. Detections under DC E-field are also studied. Without the negative potential to remove the non-specific binding, DC E-field only has the low SBR around 3.0 for RNA. In addition, DC E-field could not generate good mixing for protein binding. The highest SBR is only around 3.5 for IL-8 protein binding. With the current saliva sample and EC sensor setup, the optimized condition for both RNA and protein detection in saliva is 20 cycles of 9s at −300 mV and 1s at +200 mV (200 seconds total). However, for each specific clinical sample (serum, urine, saliva, et. al.), because of the different properties of targeting molecule and supporting media, the csw E-field condition needs to be optimized independently.
SBR with different reaction time under csw E-field and DC E-field assisted bio-sensing with (a) 5 nM IL-8 IVT RNA and (b) 12.5 ng/ml IL-8 protein standard. Mean value and standard deviation are illustrated with triplet experiment.
Sensitivity validation of EC sensor
With the csw E-field, only single optimized condition is required for both mRNA and protein with highly sensitive/specific detection. shows the calibration curve of IL-8 mRNA and IL-8 protein by spiking the standard in vitro translated (IVT) RNA and protein into saliva under multiplexing mode. The sensitivity is 3.9 fM for mRNA and 7.4 pg/ml for protein with the cutoff at 2 standard deviations. The linearity for the two species are R2=0.98 and R2=1, respectively. These results show the high sensitivity for multiplex salivary biomarker detection. In addition, the dynamic range showed in covers about 4 orders of magnitude. For IL-8 mRNA, the dynamic range covers from 5 fM to 50 pM (the mRNA concentration profile reach to the plateau at 50 pM level). For IL-8 protein, the dynamic range covers from 10 pg/ml to 12, 500 pg/ml. The wide dynamic range provides a powerful tool for variant saliva sample.
Sensitivity of EC sensor for multiplexing detection of salivary biomarkers under csw E-field with linear fit. (a) IL-8 mRNA (R2=0.98). (b) IL-8 protein (R2=1). Mean value and standard deviation are both illustrated with triplet experiments.
Multiplexed assay of salivary protein and mRNA biomarkers by EC sensor
Totally 56 saliva samples including 28 oral cancer samples and 28 control samples from India were measured using the EC sensor. For each saliva sample, the whole measurement with EC sensor is within 10 min after loading the saliva, and 100 μL of saliva are required for mRNA and protein detection together.
The EC sensor readouts are shown in with oral cancer samples in the upper panel and control samples in the lower panel. IL-8 mRNA and IL-8 protein signal are listed together for each sample. Overall, each biomarkers shows higher level of the cancer than those of the control samples. Statistical analysis showed a significant difference for both IL-8 mRNA (W=810, p
) and IL-8 protein (W = 820, p
), estimated using the Wilcoxon signed ranks test(29
). For the sample size requirements, at α=0.05, β=0.2, sample size required is 13 for each group. Therefore 28 sample size is sufficiently powered.
Figure 4 Multiplex detection of IL-8 mRNA and IL-8 protein with EC sensor of 56 saliva samples. (a) Results from cancer samples and (b) results from control samples and statistical analysis for clinical saliva samples tested by EC sensors. Box of standard deviation (more ...)
An equal number of age- and sex-matched subjects with comparable chewing, smoking and alcohol histories were selected as a control group. Among the two subject groups, there were no significant differences in terms of mean age: OSCC patients, 46 ± 12.8 years; normal subjects, 44.9 ± 11.9 years (Wilcoxon rank sum test, P > 0.8); gender (P =1); chewing history (P >0.08); smoking history (P > 0.27); or alcohol drinking history (P >0.06). All of the subjects signed the institutional review board-approved consent form agreeing to serve as saliva donors for the experiments ().
Statistical evaluation of correlation between oral cancer and control subjects
The mean value of IL-8 mRNA is −345 nA with cancer and −116 nA with control (). The mean value of IL-8 protein is −180 nA with cancer and −66 nA with control (). However, the broad distribution of two biomarkers should be noticed as well. Despite the wide spread in the of EC values for the markers in the OSCC group we can see from the box plots that there is very little overlap between the groups. For example for IL-8 mRNA 75% of the OSCC cases have lower EC values than the minimum EC value for the controls.
Receiver operating characteristic (ROC) analysis provides a good way to evaluate the accuracy in diagnostic decision making(9
). The ROC curves of the EC sensor for both IL-8 mRNA and IL-8 protein are illustrated in . The area-under-curve (AUC) is 0.90 for IL-8 mRNA and 0.91 for IL-8 protein. From the results measured with the same saliva samples by PCR and ELISA, the AUC is 0.91 and 0.87, respectively. The ROC curve analysis showed that the EC sensor for IL-8 mRNA achieved 83% sensitivity (percentage of positive samples correctly identified) and 87% specificity (percentage of negative samples correctly identified). For the IL-8 protein the EC sensor achieved 87% sensitivity and 87% specificity. These results indicate that the EC sensor is an accurate method for measuring salivary biomarkers as good as the conventional PCR and ELISA. It also demonstrates the ability to identify oral cancer subjects using a saliva-based screening.
Logistical regression has also been carried out with combined IL-8 mRNA and protein. The resultant data shows the combined IL-8 mRNA and protein did increase AUC to 0.93. The comparison between individual IL-8 mRNA, IL-8 Protein and combinational IL-8 mRNA+Protein are all illustrated in . The improvement in ROC indicates the combined IL-8 mRNA and protein further increases in area under curve (AUC).