Introduction

One of the critical factors in the diagnosis and management of diseases such as sepsis is the rapid and reliable detection of protein biomarkers in biological samples1,2. C-reactive protein (CRP) is one of the acute-phase proteins produced in the liver and is one of the most common biomarkers of infection or an inflammatory response1,3. In various acute or chronic infections, tissue damage, malignant tumors, myocardial infarction, surgical trauma, radiation damage, etc., CRP rapidly rises within several hours and returns to normal upon recovery from the disease4,5. Many methods have been developed for measuring CRP, such as ELISA6,7 immunoturbidimetry, and the electrochemical method8,9. However, the problems with these methods include their increased need for time, labor, complexity in steps and preparation, and expensive reagents8.

Among the methods that have been introduced to provide simplicity and low cost for the detection of CRP in biological samples that can simultaneously have high sensitivity and selectivity is the fluorescence resonance energy transfer (FRET) technique. This technique is very promising among the various technologies for advancing the detection power of biomarkers10. FRET occurs when an excited fluorophore or donor molecule transfers energy to an acceptor molecule via intermolecular dipole-dipole interactions11,12. During this process, donor fluorophores are excited followed by emission spectrum overlaps that of acceptors in very close range12,13. Meanwhile, fluorescent aptamers can be used as biological receptors. According to this principle, structural changes in the aptamer that can be followed by fluorescence measurement can be attributed to the amount of analyte in the sample14,15.

Aptamers (single-stranded DNA or RNA oligonucleotides16 have prominent advantages in detecting small molecular biological targets due to their high stability, low cost, simply synthesis, easy marking, no immunity source and toxicity17,18,19. By modifying an aptamer with a fluorescent probe, a suitable distance can be created between acceptor and donor molecules. In optical sensors, fluorescently tagged aptamers can be used to detect biomolecules20. The use of aptamers instead of antibodies in the antigen-detector system has advantages such as reduced cost, ease of fabrication, high affinity, and sensitivity, and can be used for a wide range of analytes21.

Previous studies on CRP detection have made significant advancements but also faced notable limitations1,22,23,24,25. For instance, the Cu-MOF-based dual-mode aptasensor22 provides flexibility with fluorescence and colorimetric detection but has higher detection limits and increased complexity. Similarly, the capillary-based ELISA system23 is portable and cost-effective for point-of-care testing but lacks the sensitivity required for early-stage diagnostics. The N-GQD and AuNP-based FRET aptasensor24 has rapid detection but requires complex synthesis and a higher detection limit. While the microfluidic-integrated capacitive biosensor25 enables label-free, real-time monitoring, its complex fabrication and moderate sensitivity limit its practicality. Finally, the RNase H-assisted DNA recycling aptasensor1 achieves signal amplification for improved sensitivity but adds complexity with enzymatic steps, limiting its simplicity for routine applications. These studies highlight the need for a simple, highly sensitive, and easy-to-fabricate platform, which is addressed by the FRET-based fluorescent aptasensor developed in this work.

Graphene oxide (GO), which has surface oxygen-containing groups, has unique capabilities that enable it to be widely used in the field of sensing. These properties include high surface area, large and easy surface modification, water dispersion, and strong luminescence26,27. Further, another capability of GO, which is due to the increase in absorption cross-section and non-radioactive excitation energy transfer ability, is that it can quench various fluorophores with high efficiency28. Hence, with these excellent properties, it can be used in the development of FRET sensors29,30,31,32. In a fluorescent sensor, the binding of the labeled aptamer to GO was established by inducing π-π stacking, and GO led to FRET33,34. It is noteworthy that the ability of GO to interact with a wide range of biomacromolecules, including proteins, peptides, and amino acids, is also exploited through this quenching process35.

Following our recent studies on the design and development of biosensors36,37,38,39,40,41, in this study, we developed a simple fluorescent sensing platform using GO as a quencher for CRP detection (Fig. 1). This platform is based on FRET using 6-carboxyfluorescein-tagged aptamer (FAM-aptamer) as the donor. The fluorescence of the dye was effectively quenched when the dye-labeled aptamer was adsorbed onto GO via π − π stacking interactions. Upon the introduction of CRP, the fluorescence was recovered as the FAM-labeled aptamer preferentially bound to CRP. This homogeneous fluorescent assay eliminates the need for complicated surface modification and probe immobilization, addressing the complexity and fabrication challenges of previous methods. Furthermore, it exhibited a high specificity towards CRP with an excellent detection limit, making it a promising tool for ultra-sensitive biomarker detection and early disease diagnostics.

Fig. 1
Fig. 1
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Schematic illustration of stages of preparation and fabrication of a fluorescence nanobiosensor for CRP detection in blood samples.

Materials and methods

Chemicals and apparatus

The CRP FAM-labeled aptamer (5′-FAM-GGC AGG AAG ACA AAC ATA TAA TTG AGA TCG TTT GAT GAC TTT GTA AGA GTG TGG AAT GGT CTG TGG TGC TGT-3′)39 was synthesized by Metabion (Germany). Graphene oxide, phosphate buffered saline (PBS), bovine serum albumin (BSA), and all other mentioned chemicals and solvents were purchased from Sigma-Aldrich. CRP was obtained from Mybiosource company (MBS390133). TNF- was obtained from Prospec company (cyt_223_b). Hemoglobin with up to 95% purity was prepared according to the method of Williams and Tsay42. Herceptin was obtained from Sinaclon (Tehran, Iran). Human serum samples were obtained from a Fardis biological analysis laboratory (Alborz, Iran). All procedures complied with relevant regulations (including the Declaration of Helsinki). Anonymized leftover human serum samples were obtained from a local diagnostic lab after routine testing, with no access to personal data. Their use was permitted under institutional policies without requiring ethics approval.

A Philips diffractometer with mono chromatized Co kα radiation (X’pert Corporation, Philips, The Netherlands) was used for XRD (X-ray powder diffraction) of the GO samples. To characterize the morphology, TEM images were taken on a Tecnai G2 F20 (Philips, The Netherlands) with an accelerating voltage of 150 kV. A NanoDrop 2000 (Thermo, USA) was used to observe and record the UV-visible absorption spectra of the labeled aptamers and their binding to GO. A fluorescence spectrophotometer F-7000 (Hitachi Corporation, Tokyo, Japan) was used to record the fluorescence spectra. The sample was placed in a 300-µL quartz cuvette, and the luminescence intensity was measured by exciting the sample at 450 nm and recording the emission at 520 nm, corresponding to the characteristic peak wavelength of FAM.

Synthesis of graphene oxide

The method used for the synthesis of GO is called the Hummers and Offeman method. In this method, natural graphite powder was oxidized and exfoliated to synthesize GO sheets43. The XRD pattern, UV − vis spectrum, and TEM image were used to characterize the synthesized GO nanosheets. The XRD pattern (Fig. S1a) shows a prominent peak at 2θ = 11.6°, corresponding to a d-spacing of 8.84 Å and the (001) reflection, attributed to oxygen-containing functional groups. Moreover, a maximum peak at 230 nm (related to the π − π* transition of aromatic C = C) and a shoulder peak at 290 nm (related to n − π* transition of C = O) in UV-vis spectrum are observed (Fig. S1b). GO nanosheets with a high transparency is also obvious in TEM image (Fig. S1c). All are indicative for the successful synthesis of GO sheets (Figs. S1a-c)44.

Fluorescent assay of CRP protein

To obtain a homogeneous brown solution of GO (4 mg/ml), GO powder was first dissolved in Milli-Q purified water and then dispersed by ultrasonic waves. Also, the stock aptamer solution (100 µM) was prepared with ultrapure DI water and stored at −20 °C before use. In sensing processing, the quenching rate, recovery efficiency, and detection sensitivity are affected by GO concentration and incubation times. Hence, the optimization of GO concentration (0–0.09.09 mg/mL) and quenching/recovery time (0–20 min) was conducted.

In order to detect CRP, at first, 1 µL FAM-aptamer (330 nM) and 0.5 µL GO solution (0.03 mg/ml) were mixed and diluted with Milli-Q purified water up to 300 µL. For sufficient interaction of the aptamers, complete dispersion is important. Hence, different concentrations of CRP were added to the FAM-aptamer-GO with gentle shaking. They were then incubated at room temperature (5 min). Then, the fluorescence spectra of the samples were recorded to check the amount of fluorescence recovered from FAM. They were also used to make the standard curve under the excitation and emission wavelengths of 450 and 520 nm, respectively. Each experiment was performed three times under optimized sensing conditions to ensure accuracy and reliability.

Results and discussion

Fluorescent aptasensor principle based on GO for CRP detection

In this study, a fluorescent aptasensor was designed using GO and FAM-aptamer for CRP detection. The entire process of the GO-based fluorescence aptasensor for CRP detection is illustrated in Fig. 2. The FRET process occurs when GO quenches the FAM fluorescence due to the proximity of the energy transfer. This phenomenon occurs in the absence of CRP due to the hydrophobic and π-π stacking of aptamers on the GO surface45,46.. In the presence of CRP protein, due to the stronger binding of the protein to the aptamers, they dissociate from the GO and bind to CRP, causing fluorescence recovery. Therefore, the intensity of FAM fluorescence recovery is indicative of the amount of target protein45,46,47,48..

As shown in Fig. 2a and b, CRP is a non-fluorescent protein and GO does not any fluorescence intensity. FAM presents high fluorescence intensity in the fluorescence spectrum of FAM-aptamer. However, upon GO addition, fluorescence intensity was remarkably reduced46. This indicates that by adsorbing together with an aptamer, GO efficiently quenched fluorescence (fluorescence ‘OFF’)45,46,47,48 (Fig. 2a). However, when CRP was added, the quenched fluorescence recovered in time (fluorescence ‘ON’) (Fig. 2a). In reality, CRP binds strongly to its aptamer compared to GO. In this case, fluorescence is restored since FAM-aptamers are located away from GO surfaces and energy transfer efficiency is decreased. In different conditions, fluorescence emission spectra of FAM-labeled aptamers and CRPs were analyzed statistically (Fig. 2b).

FRET phenomenon is based on overlapping between the emission of a donor fluorophore with the excitation and/or absorption spectrum of an acceptor when they are in very close proximity12,13. Figure 2c demonstrates the spectral overlap between the donor emission (FAM fluorophore) and GO acceptor absorption. This indicates that when FAM-aptamers are adsorbed onto the GO surface, GO efficiently quenches the fluorescence of FAM via the FRET process, resulting in a significant decrease in the fluorescence intensity of FAM due to resonance energy transfer. This stady also investigated the effect of graphen oxide on the absorption of FAM-aptamer in GO-apt fluorescent aptasensor for CRP detection. According to Fig. 2d, FAM-aptamer shows an absorption peak was at 450 nm. As seen, the FAM-aptamer’s peak absorption decreased after adding GO. This means that because of the energy transfers between GO and FAM-aptamer, the overall absorption of FAM-aptamer decreases.

Fig. 2
Fig. 2
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Evaluation of Go-apt CRP detection. (a) The fluorescence emission spectrum of FAM-aptamer (330 nM) in the presence of GO (0.03 mg/ml) with an excitation 450 nm and (b) Fluorescence emission of FAM-aptamer and CRP under different conditions: (a) FAM-aptamer, (b) FAM-aptamer + GO, (c) FAM-aptamer + GO + CRP (d) GO and (e) CRP. (c) Absorption (blue) and emission (red) spectra of acceptor (graphene oxide) and donor (FAM fluorophore) molecules and their overlap with each other (yellow). Excitation wavelength was 450 nm. (d) Absorption spectra of FAM-aptamer in the presence of GO and CRP protein.

Optimization of experimental conditions

The GO was optimized to increase the sensitivity of the fluorescent aptasensor to detect CRP protein. GO concentrations were varied between 0 and 0.09 mg/ml with aptamer concentrations fixed at 330 nM during optimization. Figure 3a shows that different GO concentrations have a significant impact on the quenching of FAM-aptamer fluorescence and also affect the fluorescence recovery of FAM-aptamer. Moreover, Fig. 3b shows the CRP effect on the fluorescence intensity of FAM-aptamer at different GO concentrations. A significant reduction in fluorescence signal background occurs after adding GO. From Fig. 3a and b, accordingly, at 0.03 mg/ml GO concentration, the ratio (F/F0)/F0 gets the highest value, which is 1.54, when F0 = FAM fluorescence intensity at 520 nm without CRP and F = FAM fluorescence intensity with CRP. It was therefore determined that 0.03 mg/ml of GO was the optimum concentration.

In order to determine the assay time, we investigated whether the fluorescence quenching and recovery times affected the assay time, as shown in Fig. 3c and d, respectively. Fluorescence intensities in quenching and recovery were measured at 0, 2, 4, 6, 8, 10, 12, 15 and 10 min. As shown in Fig. 3c, increasing incubation time increased the fluorescence quenching of FAM-aptamer, which indicated that it was adsorbing to the GO surface. As seen, quenching of FAM-apt fluorescence by GO is a very fast process, so that in the first 10 min of doing the process, significant changes were observed in the fluorescence quenching process. Therefore, minimal quenched fluorescence can be achieved within 10 min (Fig. 3c), whereas maximal recovered fluorescence can be obtained within 5 min (Fig. 3d). Therefore, a quenching time of 10 min and a recovery time of 5 min were used. A time-dependent experiment shows that GO is a wonderful quencher of FAM-apt fluorescence, rapidly quenching it (Turn-off) and regaining it (Turn-on) when CRP is present.

Fig. 3
Fig. 3
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Optimal conditions in experiments. (a) Optimal amount of GO in the presence and absence of CRP causes recovery and quenching of fluorescence intensity, respectively. (b) The fluorescence intensity rate (F/F0) of FAM-apt by CRP protein as a function of GO concentration. Excitation was 450 nm. A concentration of 330 nM of aptamer was fixed. Error bars were calculated from three independent experiments using a fixed concentration of C-reactive protein (CRP) at various concentrations of graphene oxide. (c) Kinetic behaviors of FAM-labeled aptame quenching at different incubation times with 0.03 mg/mL GO and 330 nM FAM-aptamer (emission wavelength of 520 nm). (d) Time-dependent fluorescence regeneration of FAM-apt in GO suspension by CRP. Error bars were obtained from three experiments.

Turn-on CRP detection with a fluorescent aptasensor based on GO

The optimized conditions were used to ensure the accuracy of the experimental results for CRP detection. Figure 4a shows that as the increasing CRP concentration from 33 to 274 fg/ml, the fluorescence intensity of FAM-aptamer/GO is enhanced in accordance. Additionally, the ratio of (F0-F0)/F0 shows a linear correlation with the CRP concentration in the range of 33 to 82 fg/ml for the first slope and 114 to 207 fg/mlهfor the second slope (Fig. 4b). The linear regression equation is y = 0.0561x − 1.4606 where x is the CRP concentration (fg/ml) with the regression coefficient R2 = 0.99 for the first slope and y = 0.0201x + 0.925 with the regression coefficient R2 = 0.98هfor the second slope. The limit of detection was obtained 2.27 fg/ml using the formula LOD = 3 standard deviation of the blank (0.0425)/slope calibration curve (0.0561). Hence, a wide detection range was observed using the GO-based fluorescence aptamer assay.

The comparison in Table 1 highlights the superior performance of our FAM-aptamer/GO-based fluorescent aptasensor for CRP detection in terms of sensitivity and simplicity. Compared to other fluorescence-based methods, such as RNA apt/Au NPs@Si MSs (LOD: 10 pg/mL,1) and N-GQDs/AuNPs (LOD: 20 pg/mL24,), our approach achieves an unprecedentedly low detection limit of 2.27 fg/mL. While Cu-MOF-based dual-mode sensors22 and capillary-based ELISA23provide alternative detection strategies, they suffer from higher detection limits and, in some cases, lack real-sample applicability. Electrochemical methods, such as Ti3C2Tx-Ag/Au NPs/DNA apt (LOD: 41 pg/mL49,) and AuNPs/GO-COOH/DNA apt (LOD: 1 pg/mL50,), offer robust platforms but cannot match the sensitivity of our method. Additionally, while lateral flow assays51 and colorimetric approaches39 provide broader linear ranges, they exhibit higher detection limits, making them less suitable for ultra-sensitive applications. Overall, our FRET-based aptasensor not only outperforms existing methods in sensitivity but also offers the advantage of a simplified, homogeneous assay without requiring complex modifications, making it a promising tool for early-stage CRP detection in clinical diagnostics.

Moreover, as illustrated in Table 1, in compared with the other methods, this developed sensing system has shown at least 1000 times higher sensitivity. Really, our designed fluorescent probe demonstrates a substantial improvement in detection limits of 1000 times (femtogram per milliliter) compared to previous studies. While other studies have identified biomarkers at picograms per milliliter concentrations, our sensor can successfully detect femtograms per milliliter of the biomarker, exhibiting at least a thousand-fold enhancement in detection capability.

Fig. 4
Fig. 4
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Go-apt CRP detection. (a) The GO-apt aptasensor’s fluorescence emission spectrum based on CRP concentration ranges from 33 fg/ml to 274 fg/ml. (b) Fluorescence intensity ratio (F/F0) as a linear relationship versus CRP concentration. An expression of F/F0 (where F0 and F are the FAM fluorescence intensities at 520 nm without and with CRP). (c) CRP aptasensor specificity. In the presence of CRP, BSA, TNF- , Hb, Herceptin proteins (0.01 ng/ml), the fluorescence intensity rate (F-F0)/F0 of a fluorescent aptasensor based on GO was measured. Where at 520 nm, F0 and F are the fluorescence intensities without and with CRP detection (excitation: 450 nm).

Table 1 The comparison between our method and other reported biosensors for the CRP detection.
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Specificity plays a key role in the practical application of aptamer functionalization of the GO sensor. The selectivity of the aptamer for CRP is evaluated by the fluorescence intensity response in the presence of several different proteins, such as BSA, Hb, herceptin and TNF- (0.01 ng/ml) under the same experimental conditions. As shown in Fig. 4c, the fluorescence intensity in the sample containing CRP is higher than other control groups. These results clearly demonstrate the great specificity of the suggested CRP detection strategy. This significant advancement can be attributed to the combined effects of FRET and aptamer technology, which enables selective detection while minimizing interference from the environmental matrix, ultimately resulting in superior detection levels.

Detection of CRP in real sample

To investigate the method’s recovery, our aptasensor was tested on three human serum samples collected from a local biomedical analysis laboratory. At first, 1 µl serum samples after dilution was added to the GO quenching system connected to the aptamer-fluorophore. Then, different CRP concentrations (16.63–82.64 fg/ml) were added to the designed aptasensor system on serum samples. The available CRP concentration was calculated using the measurement standard addition method. The recovery percentage was calculated as the ratio of the CRP concentration found to the added concentration. Three replicates of each concentration were tested. According to Table 2, the obtained recovery for CRP measurements was between 88.3% and 111%, showing that the proposed method for CRP detection in complex biological samples was reliable.

In summary, it can be said that, the developed GO-based FRET aptasensor provides a simple, rapid, and low-volume platform for CRP detection, achieving femtogram-level sensitivity and a wide dynamic range. This allows reliable quantification from baseline levels (~ 3 mg/L) to highly elevated concentrations (> 500 mg/L) in severe inflammation3. While such ultrasensitivity is not strictly required for routine CRP diagnostics in serum, it substantially expands the platform’s applicability to contexts where biomarker concentrations are much lower. Its ultrasensitive performance is particularly relevant for detecting CRP in low-concentration biological fluids such as saliva (285 pg/µL54; 0.05–64.3 ng/mL55), sweat (~ 970.83 pg/mL56), and tears (~ 30 ng/µL57), where conventional assays may lack sufficient sensitivity. Beyond CRP, the sensor’s design offers potential for detecting other clinically important biomarkers present at trace levels, including cancer-related proteins. The development of similar ultrasensitive CRP sensors, such as the impedimetric immunosensor reported by Kanyong et al58., further underscores the growing interest in high-performance biosensors for this critical biomarker. Moreover, compared with conventional ELISA, which typically reaches nanogram-per-milliliter detection limits59, the proposed GO-FRET aptasensor demonstrates several orders of magnitude higher sensitivity. Its performance remains robust even in highly diluted plasma, minimizing matrix effects60. Plasma dilution was deliberately applied to reduce interference from abundant serum proteins, which can otherwise mask weak fluorescence signals61, thereby making femtogram-level sensitivity essential for accurate quantification under these conditions. At the same time, the assay requires only minimal sample volume (1 µL) and provides short assay times (5–10 min). Taken together, these features highlight the aptasensor’s practical value, analytical competitiveness, and versatility, positioning it as a promising tool for routine CRP diagnostics and broader trace-level biomarker detection in both clinical and research settings.

Table 2 Determination of CRP in human serum samples after Dilution (n = 3) with GO-aptasensors.
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Conclusions

In this study, we successfully developed a cost-effective, highly sensitive, and selective fluorescent aptasensor for CRP detection. The innovative design is based on the non-covalent π-π stacking interactions between graphene oxide (GO) and FAM-labeled aptamers resulting in the exceptional fluorescence-quenching properties of GO. Using this approach, protein presence can be quantified by observing the fluorescence emitted from the aptamer-attached fluorophore upon its release from graphene oxide following protein interaction. Under optimized conditions, the aptasensor demonstrated an impressive linear detection range and an ultra-low detection limit, at least 1000 times lower than many existing methods. Its high sensitivity supports CRP detection in low-concentration biological fluids such as saliva, sweat, and tear fluid, extending its applicability beyond serum measurements. This platform offers rapid CRP detection and requires minimal real sample volume (1 µl), further enhancing its practicality. Moreover, the developed biosensor eliminates the need for complex surface functionalization, significantly simplifying fabrication and analysis. With its excellent sensitivity, selectivity, rapid assay time, and simplicity, this aptasensor shows great promise as a robust tool for CRP detection in clinical laboratories and broader biosensing applications requiring trace-level biomarker detection.