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Long noncoding RNAs (lncRNAs) are promising biomarkers for the noninvasive diagnosis of colorectal cancer, but detection of cancer-associated changes in their expression levels is now restricted to the use of RT-qPCR. To extend the clinical applicability of these biomarkers, we developed an electrochemical platform, integrated by two different sandwich genosensors, for the relative quantification of a lncRNA called colon cancer associated transcript-1 (CCAT1) with respect to the mRNA GAPDH as an endogenous control. To achieve highly sensitive detection, and in contrast to previous sensors of this class, we use multiple fluorescein-tagged hybridization assistant probes allowing the incorporation of multiple redox enzymes per target molecule, in turn favoring the selective capture of these highly-structured targets onto the sensing platform. The resultant biosensors show high sensitivity (9.7 ± 0.3 µA nM−1 for CCAT1 and 7.3 ± 0.1 µA nM−1 for GAPDH) and low limits of detection (990 fM and 1830 fM for CCAT1 and GAPDH, respectively). Coupled to a PCR assay, we detect the lncRNA at clinically relevant concentrations. Preliminary results using total RNA extracted from the colorectal cancer cell line HT29 and spiked plasma samples from healthy individuals suggest that this new sensing architecture is sensitive and selective enough to obtain the expression level of CCAT1 simply from the ratio of outputs from the two platform sensors. We envision that the same design could be applied to detect other lncRNAs associated to various malignancies, facilitating early cancer diagnosis. [Display omitted] • Hybridization sensors with multiple detection probes to favor capture of lncRNA. • Chronoamperometric platform with two genosensors for the relative quantification of CCAT1. • Expression level of CCAT1 in plasma from the ratio between the signal of the two genosensors. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) are promising biomarkers for the noninvasive diagnosis of colorectal cancer, but detection of cancer-associated changes in their expression levels is now restricted to the use of RT-qPCR. To extend the clinical applicability of these biomarkers, we developed an electrochemical platform, integrated by two different sandwich genosensors, for the relative quantification of a lncRNA called colon cancer associated transcript-1 (CCAT1) with respect to the mRNA GAPDH as an endogenous control. To achieve highly sensitive detection, and in contrast to previous sensors of this class, we use multiple fluorescein-tagged hybridization assistant probes allowing the incorporation of multiple redox enzymes per target molecule, in turn favoring the selective capture of these highly-structured targets onto the sensing platform. The resultant biosensors show high sensitivity (9.7 ± 0.3 µA nM−1 for CCAT1 and 7.3 ± 0.1 µA nM−1 for GAPDH) and low limits of detection (990 fM and 1830 fM for CCAT1 and GAPDH, respectively). Coupled to a PCR assay, we detect the lncRNA at clinically relevant concentrations. Preliminary results using total RNA extracted from the colorectal cancer cell line HT29 and spiked plasma samples from healthy individuals suggest that this new sensing architecture is sensitive and selective enough to obtain the expression level of CCAT1 simply from the ratio of outputs from the two platform sensors. We envision that the same design could be applied to detect other lncRNAs associated to various malignancies, facilitating early cancer diagnosis. [Display omitted] • Hybridization sensors with multiple detection probes to favor capture of lncRNA. • Chronoamperometric platform with two genosensors for the relative quantification of CCAT1. • Expression level of CCAT1 in plasma from the ratio between the signal of the two genosensors. [ABSTRACT FROM AUTHOR]

The prostate specific antigen (PSA) test is the gold standard for the screening of prostate cancer (PCa), despite its limited clinical specificity. Long noncoding RNAs are released from the tumor tissue to the urine and show great potential for improving specificity in PCa diagnosis. This work reports on a sandwich-type hybridization assay to detect both the urinary biomarker prostate cancer antigen 3 (PCA3) and an endogenous control, the PSA mRNA. Multiple fluorescein-tagged hybridization assistant probes are used to promote the selective capture of this long noncoding RNA, and sensitivity by incorporating multiple redox enzymes per target molecule, after addition of antifluorescein Fab fragment-peroxidase conjugate. This strategy alleviates the problems associated with the low natural abundance of this marker, its large size, and complex secondary structure. The individual genosensors exhibit good sensitivity (2.48 ± 0.01 μA nM−1 and 6.4 ± 0.3 μA nM−1 for PCA3 and PSA, respectively), with wide linear ranges (from 25 pM to 10 nM for PCA3 and 1 nM for PSA), and detection limits in the low picomolar range (4.4 pM and 1.5 pM for PCA3 and PSA, respectively). This analytical performance is retained in the dual configuration without significant cross-talk, despite using the same enzyme label. The usefulness of this dual platform was demonstrated by analyzing RNA extracts from the prostate cancer cell line LNCaP and from urine samples of prostate cancer patients. [Display omitted] • A sandwich sensor for the detection of PCA3, a lncRNA specific of prostate cancer. • Simultaneous measurement of PCA3 and an internal control in a dual electrochemical platform. • Multiple fluorescein-labeled hybridization probes to amplify the chronoamperometric reading. • A new way of screening prostate cancer in urine samples. [ABSTRACT FROM AUTHOR]