Signal-On Fluorescence Biosensor for Detection of miRNA-21 Based on ROX labeled Specific Stem-Loop Probe

Background The abnormal expression of microRNA (miRNA) influences RNA transcription and protein translation, leading to tumor progression and metastasis. Today, reliably identifying aberrant miRNA expression remains challenging, especially when employing quick, simple, and portable detection methods. Objectives This study aimed to diagnose and detect the miR-21 biomarker with high sensitivity and specificity. Methods Our detection approach involves immobilizing ROX dye-labeled single-stranded DNA probes (ROX-labeled ssDNA) onto MWCNTs to detect target miRNA-21. Initially, adsorbing ROX-labeled ssDNA onto MWCNTs causes fluorescence quenching of ROX. Subsequently, introducing its complementary DNA (cDNA) forms double-stranded DNA (dsDNA), which results in the desorption and release from MWCNTs, thus restoring ROX fluorescence. Results The study examined changes in fluorescence intensities before and after hybridization with miRNA-21. The fluorescence emission intensities responded linearly to increases in miR-21 concentration from 10-9 to 3.2 × 10-6 M. The developed fluorescence sensor exhibited a detection limit of 1.12 × 10-9 M. Conclusions This work demonstrates that using a nano-biosensor based on carbon nanotubes offers a highly sensitive method for the early detection of colorectal cancer (CRC), supplementing existing techniques.

methods have been developed, including sigmoidoscopy, CT colonography (CTC), colonoscopy, the fecal occult blood test (FOBT), stool DNA test, double-contrast barium enema, and colonoscopy (7).FOBT, which tests for hemoglobin in feces using an antibody, is the most commonly used and cost-effective method but suffers from a high rate of false positives and negatives and limited sensitivity.In contrast, CTC, sigmoidoscopy, and colonoscopy offer more accurate direct visualization of lesions but require thorough bowel preparation, are more costly, and have lower participation rates (8,9).Increasing research suggests that tumor markers, which can be proteins, enzymes, genes, gene products, specific cells, or hormones, might be detected in bodily fluids or tissues, indicating the presence of cancer.On the other hand, methods such as (enzyme-linked immunosorbent assay (ELISA) (10), immunohistochemistry (11), IHC (11), radioimmunoassay (12), fluorescence (13), chemiluminescence (14), electrophoresis (15), and polymerase chain reaction (PCR) (16) have been developed for CRC detection.While these approaches can yield reliable results, they come with several disadvantages, such as lengthy processes, complex operation procedures, and a high demand for sample volume.Additionally, the trace amounts of biomarkers present during the early stages of CRC may not be detectable.Consequently, there is a need for a rapid, accurate, simple, and cost-effective method for biomarker identification to aid in the early detection and treatment of CRC.
In recent years, research has increasingly focused on circulating microRNAs (miRNA), which have been suggested as valuable diagnostic biomarkers for various types of cancer (17,18).MicroRNAs, a significant subset of small non-coding RNAs (ncRNAs), belong to a family of short (19 -25 nucleotides), single-stranded, non-coding RNAs that regulate protein synthesis by binding to the 3' UTR of target mRNAs (19).MiRNAs are considered effective biomarkers for CRC detection because they can be found in bodily fluids and exhibit high stability in the presence of RNase activity, boiling temperatures, extreme pH levels, multiple freeze/thaw cycles, and long-term storage (20).The role of miRNA as epigenetic factors in the pathogenesis of CRC has been evaluated and confirmed in numerous studies, highlighting their potential as or enzymes (30).
Numerous studies in recent decades have underscored the role of microRNAs in carcinogenesis and tumor progression (17,19,31).Among all types of non-coding RNAs (ncRNAs), microRNAs have garnered significant attention due to their frequent dysregulation in CRC (18).Incorporating cancer-associated miRNAs, specifically miRNA-21-5p, has significantly improved the diagnostic accuracy of the APC gene mutation panel in circulating cell-free tumor DNA (ctDNA) for CRC detection (32).

Objectives
A nano-biosensor for identifying miRNA-21-5p, aimed at diagnosing CRC, was developed using MWCNTs and DNA tagged with ROX.

MiRNA Isolation Method from Clinical Samples
After obtaining informed consent, 2 ml of blood was drawn from each patient and collected in tubes containing EDTA.These samples were taken from five male patients aged between 65 and 71 years, all diagnosed with stage IV CRC.The cell-free plasma was then separated from the blood by first centrifuging at 2 000 × g for 10 minutes, followed by microcentrifugation at 1 1000 × g for 3 minutes.Following the manufacturer's guidelines, 200 µl of plasma from the cancer patients was utilized to extract microRNA using the miRNeasy Serum/Plasma Kit by Qiagen.The quantity of RNA was determined by measuring the absorbance at a wavelength of 260 nm using a spectrophotometer (UV-1800, SHIMADZU).The purity of the RNA was assessed using A260/A280 ratios.

Reagents
The specific probe for the miR sequence,

Determination of LOD and LOQ
The limit of blank (LoB), limit of detection (LoD), and identified as the lowest concentration at which the analyte could not only be reliably detected but also meet some predefined criteria for bias and precision (33).

Design Strategy
The    Measurements of various fluorescence emission spectra indicated that 12 minutes is the optimal duration for duplex formation (Figure 4C).MWCNTs@Au NCs, Atto-425 miR-92a-3p 0.1 -10 0.031 (43) In this study miRNA-21 -1.12 relationship between fluorescence intensity and cDNA concentration was nonlinear, described by the equation y = -1.2031x+ 11.986 with an R 2 value of 0.7594.By examining the fluorescence response of the biosensor to cDNA and a mismatched DNA, a qualitative analysis was conducted to assess the selectivity of the miRNA sensing platform.The DNA biosensor's LOD and LOQ were determined to be 1.12 nM and 3.2 M, respectively, as shown in Table 1.

Real-Time PCR Method
The expression levels of all analyzed miRNAs were found to be significantly different between tumor and normal cells.Specifically, expression levels of miR-21 were upregulated in CRC cells by 1.5 times compared to normal cells (P < 0.05) (Figure 6C).The results obtained from real-time PCR are in agreement with those from the biosensor, mutually confirming the validity of each method.

Discussion
Biosensor technology has seen significant advancements in the detection and diagnosis of biomarkers for CRC over recent decades (44).Biosensors are generally classified into three main types: Electrochemical, mechanical, and fluorescent (45).interactions outweigh the electrostatic repulsion, the DNA will adsorb onto the MWCNT surface (Figure 1) (48,49).The biosensors technique was used to verify MWCNT nanoparticles and the MWCNT-ssDNA conjugate through SEM electron microscopy and EDX analysis.
These characterization methods visually and chemically confirmed the presence of MWCNT nanoparticles and the successful conjugation of ssDNA onto them (44).
We suggest that this biosensing platform offers several advantages.This method can be applied in complex

3 . 3 . 3 . 4 .
underlined), as well as a non-complementary sequence, '5-GTAAGGCATCTGACCGAAGGCA-3', were synthesized by Bioneer, South Korea.The oligonucleotide sequences were Iran J Pharm Res.2024; 23(1):e144368.purified using HPLC.All oligonucleotides were dissolved in deionized water to make 100 µM stock solutions and stored at -20°C.The miR-21 probes were designed to target specific sequences at the 3' end of miR-21 in humans using Oligo Analysis Software version 7.60.The probe's 5' end was labeled with the ROX dye.The nanomaterials, including multiwall carbon nanotubes (MWCNTs) with a carbon purity greater than 95% (Cat#755125), were purchased from Sigma-Aldrich, based in the US.The carboxylated MWCNTs were initially subjected to a mixture of concentrated acids (HNO 3 :H 2 SO 4 = 1:3) under ultrasonic agitation for three hours, followed by three washes with water.Optimization of the Absorption of the ROX-Labeled miR-21 Specific Probe on the Surface of MWCNTs Previous research has determined the optimal concentration for quenching fluorophore probes and creating fluorescent biosensors to be 1 mg/mL.The procedure involved combining 10 µL of the miR-21 probe (10 pM) with 15 µL of MWCNTs (1 mg/mL) in a final solution volume of 2 mL Tris-HCl (pH 7.4, 0.02 mM).The Tris-HCl solution was composed of Tris (hydroxymethyl) aminomethane, NaCl, KCl, and MgCl 2 , all dissolved in 100 ml of deionized (DI) water.The fluorescence emission was monitored at various time intervals.To identify the ideal concentration for effectively quenching the fluorescence of the probe, different concentrations of MWCNTs were mixed with 10 µL of the miR-21 probe (10 pM).The optimal time for absorption was determined through fluorescence spectrometry to assess fluorescence quenching.The formation of MWCNT-probe conjugates was verified using energy-dispersive spectroscopy (EDX) and scanning electron microscopy (SEM) with a Zeiss-DSM 960A microscope.Fluorescence spectra measurements were conducted using a varian cary eclipse fluorescence spectrophotometer.Detection of the miR-21-Specific Sequence by a miR-21 Probe-MWCNTs-Based Nanosensor The evaluation of the hybridization response involved adding the matching target DNA to the ROX-labeled probe-MWCNT conjugate after its synthesis.To accelerate the reaction time during the initial phase, the fluorescence emission intensity was chronologically monitored.Subsequently, various concentrations of complementary DNA were mixed with the MWCNT-ssDNA conjugates to find the optimal concentration for the hybridization process, and the fluorescence intensity was measured at the ideal hybridization time.The sensitivity of the nanosensor was determined by monitoring the probe fluorescence emission across 10-fold serial dilutions (from 50 pg to 3.2 M) of synthetic complementary DNA of the miR-21 probe.The negative control comprised genomic DNA from healthy control samples.
of quantification (LoQ) were established following specific protocols.The LoB was defined as the highest apparent analyte concentration observed in replicates of a blank sample containing no analyte, calculated as the mean blank plus 1.645 times the standard deviation of the blank (LoB = mean blank + 1.645(SD blank)).The LoD corresponded to the lowest concentration of the analyte that could be detected, determined as LoB plus 1.645 times the standard deviation of a low-concentration sample (LoD = LoB + 1.645 (SD low concentration sample)).Replicates of a sample known to contain a low concentration of the analyte were tested for this purpose.Finally, the LoQ was 4 Iran J Pharm Res.2024; 23(1):e144368.

3. 6 .
Detection Process Using Real-Time PCR Stem-loop real-time RT-PCR was utilized to evaluate miRNA expression.The design for miR-21 stem-loop reverse transcription (RT) primers and amplification primers followed the method outlined by Huang et al. (23).Specific stem-loop RT primers enabled the generation of cDNAs from total RNA, with the miR-21 sequence being: '5 -GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATAC GACTCAACA -3 .The reverse transcriptase reactions included 10 ng of RNA sample, 60 nM stem-loop RT primer, 1 × RT buffer, 0.25 mM of each dNTP, 4 U/µL M-MLV reverse transcriptase (Promega, Madison, WI, USA), and 0.4 U/µl RNase inhibitor (Takara).The reactions (10 µL) were incubated in a GenAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) at 16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes, with a final holding step at 4°C.Real-time PCR was conducted using a Thermal Cycler Dice real-time system TP800 (Takara).The universal reverse primer for miR-21 was '5 -CAGTGCAGGGTCCGAGGT-3 , and the specific forward primers were '5 -GCCCGCTAGCTTATCAGACTGATG-3 (miR-21).A 25 µL PCR reaction mixture containing 1× SYBR premix Ex Taq mix (Takara), 2 µL RT products, and 10 nM of each forward and reverse primer was incubated in a 96-well plate at 95°C for 30 seconds, followed by 45 cycles at 95°C for 15 seconds and 60°C for 21 seconds.A dissociation step from 65 to 95°C confirmed the specificity of the amplification products.Threshold cycle data were derived using the second derivative max settings.The U6 gene served as an internal control for normalizing the levels of the target miRNA.The stem-loop reverse transcription (RT) primers and amplification primers for U6 were sourced from Ribobio Co., Ltd., in Guangzhou, China.3.7.Statistical Analysis All fluorescence measurement experiments were conducted in triplicate on one day and across three different days.The results presented in each figure are expressed as mean values ± standard deviation.All statistical analyses were carried out using the GraphPad Prism 9 statistical program.The statistical differences among the mean fluorescence emission values of the biosensor in reaction with the control and miR-21 were determined by one-way ANOVA, with statistical significance established at P < 0.05 and P < 0.01.
fundamental concept behind the proposed fluorescent nucleic acid sensing platform is depicted in Figure 1.This concept is based on the quenching of fluorescence due to the adsorption of a fluorescently labeled single-stranded DNA (ssDNA) probe (ROX-labeled probe) onto MWCNTs.Conversely, when complementary DNA (cDNA) is introduced, a double-stranded DNA (dsDNA) forms between a ROX-labeled probe and cDNA.This duplex is released from the surface of MWCNTs, resulting in the restoration of fluorescence emission.SEM and EDX tests were employed to validate the creation of the MWCNT-ssDNA conjugates.Changes in the diameter and morphology of MWCNTs were observed in the SEM images of the MWCNTs/ssDNA probe conjugates (Figure 2A and B), indicating that the ssDNA probe was successfully adsorbed and stabilized by the MWCNTs.Energy-dispersive spectroscopy was utilized for elemental analysis.According to the results, the EDX spectrum Iran J Pharm Res.2024; 23(1):e144368.

4. 2 .
Analytical Characterization of the Designed Nanobiosensor in the Presence of miR-21 The hybridization reaction was conducted following the preparation and characterization of the MWCNT-ssDNA conjugate.Initially, the ROX-labeled probe, which emits fluorescence at 605 nm, experienced quenching upon its immobilization on the MWCNT surface.However, the fluorescence emission was restored due to complementary base pairing after the addition of the complementary sequence to the probe and the execution of the hybridization process.The analysis focused on the fluorescence emission spectra of an ssDNA probe tagged with ROX at the 5'-end, specific to the miR-21 sequence.In the absence of MWCNTs, the probe exhibited strong light emission at a wavelength of 605 nm.Nevertheless, the addition of MWCNT (40 µg) led to a significant reduction in fluorescence emission, with up 6 Iran J Pharm Res.2024; 23(1):e144368.
Figure 4A and B display the fluorescence emission spectrum in the presence of different DNA target concentrations, demonstrating how fluorescence emission intensity escalates with rising DNA target concentration.According to the analysis, the Iran J Pharm Res.2024; 23(1):e144368.

Figure 5
Figure 5 demonstrates that the biosensor's fluorescence signals in response to three-base mismatched DNA were 38.05% of those observed with the complementary DNA (cDNA).These results suggest that the nano biosensor specifically responded to the target cDNA, unlike other sequences.During the hybridization process for miRNAs isolated from the blood of cancer patients, an average fluorescence emission intensity of 142.6 was recorded, significantly higher than that of non-cancer individuals, which had an average emission intensity of 49.3 (Figure 6).The findings indicate that the presence of the probe-target sequence in blood samples from CRC patients (miRNA concentration extracted from plasma samples, 0.424 µg) resulted in significantly higher fluorescence emission in the hybridization reaction of miR-21.

Fluorescence
biosensors are non-invasive analytical tools designed to detect biomolecules in biological samples by sensing the absorption of electromagnetic radiation by fluorophores or fluorescently labeled molecules (46).These biosensors have been developed using a range of nanoparticles, including carbon, gold, and silver nanoparticles.Fluorescent sensors are highly 8 Iran J Pharm Res.2024; 23(1):e144368.

Figure 3 .Figure 4 .Figure 5 .
Figure 3. A, fluorescence emission spectrum of ROX-ssDNA in the presence of MWCNT at different time points.The intensity of fluorescence scattering decreased with increasing time, and after 6 minutes, no significant difference in fluorescence scattering intensity was observed.B, fluorescence spectrum of ROX-ssDNA in the presence of different concentrations (1 mg/mL) of MWCNT for complete extinction of fluorescence emission.As the amount of MWCNT increased, the intensity of fluorescence emission decreased, and after adding 40 µL of MWCNT nanoparticles, complete extinction of fluorescence emission was observed.
systems and avoids interference between Raleigh light scattering signals and dye fluorescence signals, thereby enhancing detection sensitivity.Consequently, it could eliminate the need for multiple laser excitation sources.Given the planar shape of MWCNTs and the simplicity of operation, the proposed method can adsorb a diverse array of DNA probes (50).Rafiee-Pour et al. developed an electrochemical biosensor capable of detecting miRNA-21 without the need for labeling, specifically aimed at identifying breast cancer (51).Salahandish et al. created an electrochemical nano-nanosensor using an NFG/AgNPs/PANI electrode combination to detect miRNA-21 cancer markers, which proved to be highly sensitive and specific (52).For detecting miRNA-21 expression in cancer cells, Liu et al. demonstrated the use of a fluorescent biosensor equipped with a 2-aminopurine (2-AP) probe alongside signal amplification (53).This biosensor amplifies the fluorescent signal in the presence of the target miRNA.Thanks to our enzyme-free signal amplification method, the sensor becomes easier and more cost-effective to use, potentially reaching a detection limit of 3.5 pM.This technique successfully identified the overexpression of miRNA-21 in human breast cancer cells.The proposed sensor could serve as a rapid and precise platform for detecting target miRNA, holding significant potential for the convenient monitoring of various miRNA biomarkers for the early detection of different cancers (53).Previous studies have indicated that miRNAs are modulated during the progression of colorectal tumors through overexpression, downregulation, or deletion (54).Wang et al. developed a fluorescent biosensor for the detection of miRNA in live cells facilitated by MnO 2 nanosheets.This method employed fluorescence resonance energy transfer (FRET) to detect miRNA-21, using FAM as the fluorescent donor and TAMRA as the fluorescent Iran J Pharm Res.2024; 23(1):e144368.

Figure 6 .
Figure 6.The fluorescence spectra and real-time PCR data were used to evaluate the applicability of the biosensor for detecting miR-21 in five different patient serum samples.Statistical analysis of the main samples is also presented.A, fluorescence spectra of the biosensor for miR-21 detection.Significant fluorescence restoration was observed in miRNA extracted from plasma samples compared to the control (normal sample) (P < 0.05).B, statistical analysis of the main samples.Non-parametric one-way ANOVA was performed for statistical analysis.Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.C, bar graphs illustrating the expression levels of miR-21 in cancer patients and controls.miR-21 was significantly up-regulated (P < 0.05) compared to normal cells.
biomarkers for the diagnosis of surgically curable stage II CRC.The main challenge with stage II CRC is the risk of disease recurrence and increased mortality.miR-21 (24)22)out as a crucial biomarker for stage II CRC and is among the microRNAs (miRNAs) extensively researched across various cancers.Studies have shown that miR-21 is significantly overexpressed in a broad spectrum of cancers, including esophageal, gastric, breast, colorectal, hepatocellular, pancreatic, as well as in glioblastoma, leukemia, B-cell lymphoma, cholangiocarcinoma, lung cancer, and squamous cell carcinomas of the cervix, tongue, neck, and prostate.This widespread elevation suggests miR-21's key role in the onset, development, and spread of many types of cancer.As an oncogenic microRNA, often referred to as "oncomiR," miR-21 plays a critical role in regulating the cell cycle, apoptosis, migration, differentiation, and stem cell renewal.Many of its targets are involved in the initiation, transformation, invasion, and metastasis of cancers(21,22).whichcannotidentify colon tumors at the molecular level(24).Nanotechnology focuses on manipulating materials at the nanoscale to detect malignant or precancerous cells 2 Iran J Pharm Res.2024; 23(1):e144368.have become essential elements in biosensor designs due to their outstanding electrical, optical, and mechanical properties (28).Defined as seamless cylindrical structures that can consist of one or several layers, either with open or closed ends, carbon nanostructures (CNTs) are categorized into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs).

Table 1 .
Comparison of Limit of Detection of miR-21 Biosensor and Other Nanobiosensors Table 1 presents a comparison of this study with others, showing that the obtained results are promising.JA and SH discussed the results; SH, JA, SHZ, and LT analyzed and interpreted the results; LT supervised, directed, and managed the study; SH, JA, SHZ, LT and SA performed the approval of the final version.Ethics code: IR.IAU.TNB.REC.1400.082.The cost of this project was borne by PhD student Somayeh Heydarian.
Data Availability:The dataset presented in the study is available on request from the corresponding author during submission or after publication.Ethical Approval: