Introduction

Sepsis, also known as bloodstream infection and blood poisoning, is a serious and potentially life-threatening infectious condition that typically occurs in the body as a result of infections by pathogens such as bacteria and viruses (Russell 2006). Sepsis is characterized by spread of the original infection site through the bloodstream to other parts of the body. Sepsis usually begins with inflammation around the site of infection. Pathogens such as bacteria or viruses can then spread through the bloodstream from the infection site to other organs or tissues, leading to exaggerated immune system responses and enhanced inflammatory reactions, which can cause inflammatory responses and tissue damage within the body (Martin et al. 2003; Riedemann et al. 2003). Sepsis can result in severe complications. It can be life-threatening in severe cases. Common symptoms of sepsis include fever, confusion, rapid breathing and heart rate, and low blood pressure. Early diagnosis and treatment are crucial for sepsis management (Remick 2007). According to a report released by the World Health Organization in 2020, sepsis occurs in more than 50 million people worldwide and causes more than 10 million deaths (Organization 2020). In South Korea, according to statistics from the Korea Centers for Disease Control and Prevention (KCDC), 5235 deaths from sepsis occurred and 25,697 patients received sepsis-related tests in 2022 (Kim et al. 2022). According to the European Society of Anaesthesiology and Intensive Care (ESAIC), sepsis accounts for approximately 50 million cases and 11 million deaths worldwide each year, representing one-fifth of all global deaths (Yébenes et al. 2017). In Europe, sepsis causes around 3.4 million cases and nearly 700,000 deaths annually, with the majority of them being preventable (Evans et al. 2021). However, it is believed that these numbers are underestimated due to the lack of accurate data collection and reporting, which remains a significant issue in sepsis management. It has been reported that sepsis can progress to septic shock when treatment is delayed, leading to rapidly increases of mortality rate by 50% (Hotchkiss et al. 2016). Therefore, prompt diagnosis and treatment are essential to increase the survival rate of sepsis patients. The most appropriate time for diagnosis and treatment is 1 h before the onset of fever due to sepsis or bacteremia. However, current diagnostic methods using blood culture tests cannot diagnose sepsis at this stage (Duncan et al. 2021). Therefore, new diagnostic methods using new materials that can quickly diagnose sepsis are needed.

Procalcitonin (PCT) is a precursor of calcitonin, one of the thyroid hormones. It is a substance that normally exists in the blood. In normal people, blood PCT concentration is 0.00 to 0.49 ng mL–111. PCT level increases in the extracellular fluid. It is produced within cells in response to inflammatory reactions (Becker et al. 2008). Sepsis is a severe inflammatory response caused by infection. Without a proper treatment, sepsis can lead to significant tissue and organ damage. PCT is considered a useful tool for early diagnosis and monitoring of sepsis. Generally, higher levels of PCT indicate a more severe infection and an increased likelihood of sepsis (Becker et al. 2010). Elevation of PCT level in sepsis patients can be used as an indicator of inflammatory responses to aid in early diagnosis and monitoring of treatment. Early diagnosis has a significant impact on treatment and outcomes of sepsis. PCT demonstrates a particularly high value in the diagnosis of sepsis due to its specific increase in sepsis compared to other inflammatory conditions (Carrol et al. 2002; Hassan et al. 2022). Therefore, it has a higher utility than other biomarkers in the diagnosis of sepsis.

Currently, methods used for early diagnosis of sepsis include clinical symptom evaluation, blood culture, imaging tests, and bronchoscopy. Clinical symptom evaluation involves assessing patients' symptoms to determine the possibility of sepsis (Zea-Vera and Ochoa 2015; Paoli et al. 2018). Blood culture is a reliable method for diagnosing sepsis, although it may take some time to obtain test results. On the other hand, imaging tests and bronchoscopy are faster, although they might have lower accuracy than the previous methods in diagnosing sepsis. To address these limitations, blood tests are primarily used. However, indicators such as white blood cell count and platelet count can be altered not only by sepsis, but also by various other causes (Liesenfeld et al. 2014; Huang et al. 2013; Chan and Gu 2011). Therefore, a detection technique that can specifically and accurately identify sepsis with greater speed and precision is needed. Thus, the aim of this study was to incorporate a new substance for the diagnosis of sepsis.

As mentioned earlier, a material called aptamer is utilized as a diagnostic tool for sepsis. Aptamers are single-stranded oligonucleotides composed of ssDNA and RNA. They can be chemically synthesized. Compared to antibodies, aptamers have advantages in terms of cost and time, which can be combined with high affinity and specificity to target substances. Additionally, antibodies have limited modifications. On the other hand, aptamers made of nucleic acids could be easily modified, enabling various studies using aptamers. The high binding specificity of aptamers is due to their ability to recognize the 3D structure of target factors. By recognizing and binding the structure of its target, an aptamer can uniquely detect target substance through structural recognition even if the same sequence exists on the surface of the target protein. Therefore, sepsis can be diagnosed early by efficiently detecting PCT even if various complex components are present in the blood (Ahn et al. 2018; Kim et al. 2009; Kim et al. 2011; Lee et al. 2015; Oh et al. 2020; Oh et al. 2022; Sekhon et al. 2017a; Sekhon et al. 2022; Sekhon et al. 2017b; Sekhon et al. 2018; Shin et al. 2022; Shin et al. 2018a; Shin et al. 2018b; Song et al. 2017; Song et al. 2018).

In this study, we developed an aptamer-based PCT detection platform for sepsis diagnosis. First, we selected candidate aptamers that could specifically bind to PCT protein. Selected candidates were quantitatively assessed for their affinities using various molecular methods. Based on these affinities, we identified the top aptamer with the highest affinity. Its binding structure with PCT was confirmed through three-dimensional structural analysis (Oh et al. 2020; Shin et al. 2022). This provided support for results of the preceding molecular experiments. To further confirm the binding of aptamers, we utilized Electrophoretic Mobility Shift Assay (EMSA) as an additional method to visually confirm the binding status of aptamers in experiments. Finally, we developed an aptamer-based PCT detection platform using aptamers selected through previous experiments (Oh et al. 2020; Shin et al. 2018a; Shin et al. 2018b). The developed PCT detection platform was evaluated for its ability to detect PCT in various types of samples.

Materials and methods

Proteins and buffer solutions for SELEX

Proteins used in this study were prepared as follows. Recombinant human PCT with a C-terminal poly-histidine tag was purchased from ABBIOTEC. (Escondido, CA, USA). Anti-PCT monoclonal antibody was obtained from Thermo Fisher Scientific. (Waltham, MA, USA). The following solutions were prepared for the SELEX technique: binding buffer (500 mM NaCl, 20 mM Tris–HCl, 25 mM imidazole, pH 7.9), wash buffer (500 mM NaCl, 20 mM Tris–HCl, 60 mM imidazole, pH 7.9), and elution buffer (300 mM NaCl, 20 mM Tris–HCl, 250 mM imidazole, pH 7.9). Phosphate-buffered saline (PBS, pH 7.2) was bought from Gibco BRL (New York, USA).

In vitro selection of aptamers by SELEX

DNA aptamer was made using a random pool consisting of 40 random nucleotides and 18 fixed sequences for primer binding (5'-ATACCAGCTTTATTCAATT (N40) AGATAGTAAGTGCAATCT-3′). Amplification of the aptamer pool was carried out through PCR with the following conditions: initial denaturation at 95 °C for 1 min; 20 cycles of 45 s of denaturation at 94 °C, 1 min of annealing at 55 °C, 1 min of elongation at 72 °C; and 5 min of elongation at 72 °C. PCR products were then purified with a PCR purification kit (Qiagen, Hilden, Germany). The aptamer pool (720 pmol) was denatured at 95 °C for 5 min and slowly cooled to room temperature to form a unique 3D structure depending on its sequence. Structured ssDNA was then reacted with human PCT protein (72 pmol) at 4 °C for 1 h. The PCT-aptamer complex was then incubated with Ni–NTA agarose (Qiagen, Hilden, Germany) for aptamer selection. The Ni–NTA agarose was pre-treated with a binding buffer. After incubation, the Ni–NTA agarose was washed several times using a washing buffer. The PCT-aptamer complex was then eluted using an elution buffer. The eluted PCT-aptamer complex was treated with PCI (Phenol:Chloroform:Isoamyl Alcohol 25:24:1) to remove PCT protein. The PCT-binding aptamer was then obtained through ethanol precipitation. Obtained aptamers were directly diluted with 100 μL of DNase-free water and used as templates for the next round of SELEX via PCR. Each round was conducted with similar procedures.

SELEX was conducted for a total of ten rounds. Between rounds 6 and 7, a negative selection round was performed using only Ni–NTA agarose without PCT-aptamer complex binding to remove sequences that could bind to Ni–NTA agarose, thus improving selectivity of the aptamer. After completing a total of ten rounds of SELEX, the concentration of the eluted aptamer pool from each round was measured using a NanoDrop spectrophotometer (Thermo Scientific, Rochester, NY, USA).

The aptamer sequence amplified with Taq polymerase was then subjected to TA cloning using a T-Blunt™ PCR Cloning kit (SolGent, Daejeon, Korea). Thereafter, competent cells (DH5α) transformed with the aptamer clone were cultured on a medium plate containing AXKI (Ampicillin 1 mg mL–1, X-gal 0.48 mg mL–1, Kanamycin 0.5 mg mL–1, and IPTG 0.25 mg mL–1 in LB). If the aptamer sequence was inserted, the lacZ gene in the cloning vector was disrupted and the X-gal contained in the AXKI medium plate could not be decomposed, leading to white colonies. However, lacZ gene was normally maintained without insertion of the aptamer sequence and blue colonies would be formed, because X-gal was decomposed to form blue degradation products (blue colonies). Under this principle, blue–white color screening was conducted to select only white colonies. Colony PCR was then performed using a Maxime PCR PreMix Kit (iNTRON Biotechnology, Seongnam, Korea) and aptamer amplification primers. After that, purified aptamer PCR products were subjected to sequence analysis (Bioneer, Daejeon, Korea). Aptamer sequence was then aligned with a Clustal X software (version 1.83).

Measurement of aptamer affinity

The binding affinity of the PCT aptamer was measured by surface plasmon resonance (SPR) using a BIAcore 3000 instrument (GE Healthcare, Chicago, IL, USA). A carboxymethylated 3D dextran matrix CM5 chip (GE Healthcare, Chicago, USA) was used for immobilizing PCT proteins. Aptamer affinity (KD) of PCT-immobilized CM5 chip was measured using the BIAcore 3000. First, PCT protein was immobilized on the surface of a CM5 chip. Protein immobilization was carried out through the following process. Prior to the PCT fixation step, 0.4 M N-(3-Didecaminopropyl)-N-side carbodiimide hydrochloride (EDC) was mixed with 0.1 M N-hydroxy sulfosuccinimide (NHS) at a 1:1 ratio. The mixture was used to rinse the sensor chip for 10 min. Then, 100 μg mL−1 PCT protein was flowed with a pH 4.5 sodium acetate solution for 7 min and fixed. Thereafter, free carboxyl groups that did not react with PCT were blocked with 1 M ethanolamine (pH 8.5) for 10 min.

Baseline signal was stabilized using HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P20). Selected 17 PCR aptamer candidates were diluted to have various concentrations (0, 10, 100, 500, and 1,000 nM). Diluted aptamers were injected into the surface of the sensor chip coated with PCT protein. The affinity between the PCT protein and each aptamer was measured in the form of a dissociation constant (KD). After measuring the affinity of one aptamer sequence, the bound aptamer was dissolved from the PCT protein fixed to the sensor chip. Sensor chip surface was regenerated until the signal reached the baseline level using a regeneration buffer (1 M NaCl, 50 mM NaOH). The exact quantification of affinity was determined with a BIA evaluation software (GE Healthcare, Chicago, IL, USA).

As an additional method to measure affinity of the obtained aptamer candidate, each aptamer was combined with the same concentration of PCT protein. After performing washing and eluting steps as described for SELEX, each aptamer was obtained using clones previously secured during the candidate selection process. Clones were added with LB broth and incubated at 37 °C for 12 h, after which bacteria were centrifuged at 3,000 rpm for 10 min. Afterward, TA vector with aptamer sequence inserted was obtained using a DNA-spin Plasmid DNA Purification Kit ((iNTRON Biotechnology, Seongnam, Korea). The aptamer sequence was amplified using PCR (Polymerase Chain Reaction) utilizing aptamer primers with the TA vector as a template. Amplified products were then purified using a PCR purification kit (Qiagen, Hilden, Germany) to obtain only aptamer sequence. Each aptamer sequence was adjusted to a concentration of 250 pmol and reacted with 50 pmol of PCT protein at 4 °C for 1 h. The PCT-aptamer complex was then incubated with Ni–NTA agarose at 4 °C for 1 h to allow binding. After undergoing a washing process, each PCT-aptamer complex was eluted using an elution buffer, followed by PCI treatment and ethanol precipitation to obtain aptamer sequence alone. The aptamer was then dissolved in 100 μL of DNase-free water. The concentration of each eluted aptamer was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Rochester, NY, USA).

Aptamer structure construction and in silico docking position confirmation

In the previous experiment, four aptamers were selected based on their highest affinity for the PCT protein. 3D docking simulation was then performed to investigate the structures of 3D binding between these aptamers and PCT protein. Before studying the structure of 3D binding between PCT protein and the aptamer, the structure of the aptamer was generated. Four PCT aptamers’ secondary structures were predicted using Mfold web server (http://mfold.albany.edu/?q=mfold) (Zuker 2010). Formation of the aptamer structure is highly dependent on salt concentration. The structure of the aptamer was formed under conditions of 250 mM [Na+] and 5 mM [Mg2+] at room temperature (25 °C). The 3D structure model of PCT protein was obtained from Alphafold database (P01258) (https://alphafold.ebi.ac.uk/) (Ruff and Pappu 2021).

The docking structure between PCT protein and the aptamer was predicted using the triangle matching method in the MOE software (MOE version 2016.0802). After minimizing root-mean-square (RMS) gradient of PCT using an energy minimization algorithm (gradient: 0.1, force field: Amber10: EHT), docking simulation was performed between PCT and the aptamer (Rabal et al. 2016). Results of the docking simulation were analyzed using PyMOL (Shin et al. 2018a) and LigPlot+ (Laskowski and Swindells 2011) software to identify hydrophobic interactions and hydrogen-bonding interactions involved in the binding between PCT and the aptamer. In LigPlot+, sequences involved in hydrophobic interactions and hydrogen-bonding interactions were identified and lengths of hydrogen bonds were determined.

Electrophoretic mobility shift assay (EMSA)

The following buffers were used for EMSA: solution A [40% acrylamide/Bis solution, 29:1 (3.3% C)] (Bio-Rad, Hercules, CA, USA), solution B (1.5 M Tris–HCl, pH8.8), solution C (0.5 M Tris–HCl, pH 6.8), and electrophoresis buffer (25 mM Tris buffer with 192 mM glycine).

A native polyacrylamide gel with 10.5% acrylamide was prepared for EMSA to visualize the 17 kDa PCT protein through electrophoresis. The mixture for the 10.5% native polyacrylamide gel was prepared using the following ingredients: solution A, 3.5 ml; solution B, 1.9 ml; N,N,N′,N′-Tetramethyl ethylenediamine (TEMED, Sigma-Aldrich, St. Louis, MO, USA), 10 µl; 10% ammonium persulfate (APS, Sigma-Aldrich, St. Louis, MO, USA), 50 µl; and distilled water, 4.6 ml. To complete preparing the native polyacrylamide gel for EMSA, the stacking gel was prepared by mixing 0.67 ml solution A, 1 ml solution C, 5 µl TEMED, 30 µl 10% APS, and 2.3 ml distilled water. The gel was then pre-run at 100 V for 30 min on ice using an electrophoresis buffer to equilibrate the buffer within the gel. After that, gel wells were washed using the electrophoresis buffer. For EMSA, the sample was prepared by incubating PCT protein and DNA aptamer at a 1:5 ratio at 4 °C for 2 h. Care was taken to ensure that the binding between the PCT protein and aptamer in the sample was not disrupted. Electrophoresis was carried out at 90 V for approximately 90 min. Subsequently, the gel was stained with EtBr to visualize the nucleic acid aptamer. EMSA results were then examined.

Aptamer-based ELISA

Selection of the optimal PCT detection aptamer

To verify the binding output of four different aptamers to PCT, a protein-binding 96-well plate (2498, Corning, Somerville, MA, USA) was treated with PCT monoclonal antibody (MA1-20,888, Thermo Fisher Scientific, Rochester, NY, USA) at a concentration of 100 ng/well and incubated at room temperature for 1 h. After that, the plate was washed with PBS-T (1X PBS with 0.1% Tween-20) three times. Next, the plate was treated with PCT protein at a concentration of 10 ng mL−1 and incubated at 4 °C for 1 h. After the incubation, the plate was washed three times with PBS-T. For PCT labeling, the plate was treated with FAM-modified aptamer (IDT, Coralville, IA, USA) at a concentration of 1 µM and incubated at room temperature for 1 h. Five wash steps were performed using the same procedure as described earlier. Once all steps were completed, fluorescence intensity of FAM was measured using a SpectraMax M2 device (Molecular device, San Jose, CA, USA) (Fig. 6A, B). PCT antibody alone (Control 1), PCT antibody with PCT protein (Control 2), and PCT antibody with FAM-modified aptamer (Control 3) were used as controls.

PCT detection range

To determine detection ranges of two selected optimal aptamers for PCT in the previous experiments, a PCT antibody-coated 96-well plate was prepared following the same procedure as before. PCT protein was then treated with aptamer at various concentrations (1–10 ng mL–1, tenfold dilution range) and incubated at 4 °C for 1 h. Subsequent steps were the same as the previous experiment. Fluorescence intensity of FAM was then measured (Figs. 6C, 6D, and 6E).

Detecting PCT in human serum

The 96-well protein-binding plate was coated with PCT antibody following the same procedure as described before. Human serum spiked with PCT protein at a concentration of 0.1 ng mL–1 was stored at 4 °C for 1 h. Subsequently, PCT detection was confirmed using FAM (Fig. 6F). All experiments were conducted under the same conditions with duplications.

Results

In vitro selection of aptamers by SELEX and affinity quantification

In this study, specific PCT-binding aptamers were isolated using SELEX aptamer screening procedure. DNA aptamer candidates against PCT protein were generated using a modified SELEX protocol. The concentration of eluted ssDNA in each selection round was monitored using a NanoDrop spectrophotometer, which reflected the binding capacity between the selected ssDNA aptamer pool and the PCT protein target. It was found that ssDNA concentrations increased in each round until the 6th round of selection, indicating enrichment of the ssDNA pool after each selection round. After a negative round, ssDNA aptamer concentrations of the 8th and 9th selection rounds were 488.4 and 491.9 ng μL−1, respectively. The concentration of the aptamer pool started to decrease from round 10 and continued to decrease until round 11. This indicated a decrease in the overall affinity of the aptamer pool (Fig. 1). Many previous studies have provided data that could support this assertion (Shin et al. 2022; Shin et al. 2018a; Shin et al. 2018b; Song et al. 2017). A total of 40 clones were selected for DNA sequencing by amplifying the PCT-binding aptamer pool. Finally, 17 different DNA sequences were identified (Table 1).

Fig. 1
figure 1

SELEX round-specific measurement of released aptamer concentration. Concentration of released ssDNA was measured using Nanodrop at each selection round in SELEX process for PCT (Procalcitonin) aptamer selection. A negative round (NR) was performed after six rounds to eliminate non-specific binding aptamers that did not directly bind to PCT. The ssDNA pool from the ninth round was selected as the optimal selection round as it showed the highest concentration of ssDNA measured

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Table 1 Sequences and SPR affinity measurements (KD) of 17 PCT aptamers
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SPR experiments were performed to quantitatively determine affinity of aptamer candidates (Fig. 4A). An SPR assay was used to analyze specific binding of 17 aptamers to PCT and determine binding affinity (KD values) of ssDNA aptamer candidates (Table 1). KD values of ssDNA aptamer candidates were measured using BIA evaluation software (GE Healthcare, Chicago, IL, USA). Of these ssDNA aptamer candidates, PCT_Ap1 to PCT_Ap17 had low KD values at the nanomole scale (Table 1).

We also conducted experiments to evaluate the binding affinity of each of the 17 aptamers with PCT. Each aptamer was bound to the PCT protein at the same concentration. After washing, concentration of the aptamer subsequently eluted could reflect its affinity toward PCT protein. Experimental results showed that concentrations for eluted aptamers of PCT_Ap1, PCT_Ap11, PCT_Ap14, and PCT_Ap17 were 511.15 ng μL–1, 607.95 ng μL–1, 578.7 ng μL–1, and 534.5 ng μL–1, respectively, which were higher than another aptamer (Fig. 2). Through this experiment, we selected these 4 out of 17 aptamers based on their eluted aptamer concentrations. Results were consistent with the previous SPR results.

Fig. 2
figure 2

Release concentrations of each aptamer post-SELEX. Concentrations of each aptamer after the post-SELEX process for the comparison of binding affinities were measured. Seventeen aptamer sequences selected through SELEX were individually combined with PCT under identical conditions. Subsequently, the same SELEX process was performed and concentrations of the released ssDNA were measured. A concentration of 500 ng μl−1 was set as the reference point. Four aptamer sequences (PCT_Ap1, PCT_Ap11, PCT_Ap14, and PCT_Ap17) showed higher release concentrations than this threshold was finally selected

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In silico 3D molecular docking simulation

An in silico 3D structural analysis was conducted to examine structures of four aptamers selected through previous research and to investigate their binding modes with PCT in detail. Using mfold and PyMOL software, 2D and 3D structures formed by each aptamer were determined based on their sequences. Each aptamer exhibited a unique structure depending on the arrangement of complementary nucleotide sequences. These aptamers showed thermodynamically stable structures with Gibbs free energy changes (ΔG) ranging from -3 kcal mol−1 to -9 kcal mol−1 (Fig. 3). Using these obtained 3D structures, docking simulations were performed with the PCT protein.

Fig. 3
figure 3

2D and 3D structure images of PCT aptamers. A–D The 2D and 3D structures of the four aptamers (PCT_Ap1, PCT_Ap11, PCT_Ap14, and PCT_Ap17) selected in the previous post-SELEX process. 2D structures were obtained using the mfold software, while the 3D structures were acquired through the PyMOL software

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Upon further analysis, the PCT_Ap1 aptamer formed three hydrogen bonds with the PCT protein. The t38 nucleotide of PCT_Ap1 formed a hydrogen bond with the Arg29 amino acid of the PCT protein. Additionally, hydrogen bonds were formed between t56-Gln56 and a16-Ser76. Apart from hydrogen bonding, a hydrophobic interaction (t13-Thr90) contributed to the binding between the aptamer and the PCT protein (Fig. 4A). When binding formations of other aptamers were examined, it was found that PCT_Ap11 formed a total of seven hydrogen bonds (t29-Lys83, t29-Asn101, g32-Thr90, g27-Arg81, c31-Cys85, c19-His104, and g21-His104) (Fig. 4B) and nine hydrophobic interactions (g27-Arg81, t65-Pro36, a40-Met92, a40-Leu88, a35-Leu93, g37-Leu93, g37-Try96, c19-His104, and g21-His104). On the other hand, PCT_Ap14 aptamer formed one hydrogen bond (a48-Lys118) and six hydrophobic interactions (g26-His104, t2-Asn101, g20-Ser89, c4-Thr90, g46-Thr105, and g46-Phe106) (Fig. 4C) in its binding configuration. Finally, PCT_Ap17 formed six hydrogen bonds (a21-Glu71, a23-Ser79, c24-Ser75, a74-Lys119, a25-Gly86, and a25-Thr90) and eight hydrophobic interactions (2 interaction sites in a23-Ser79, a23-Ser75, c24-Arg72, c56-Arg128, two interaction sites in a74-Lys119 and a73-Lys119) (Fig. 4D) as determined with LigPlot+ software. Indeed, when examining the binding positions of the aptamers to PCT, it can be observed from Fig. 4 that all four aptamers form binding structures on the same face of PCT, with PCT maintaining a consistent position.

Fig. 4
figure 4

Measurement of AFP aptamer affinity using SPR. 3D structural interaction of PCT-binding aptamers with PCT protein. A–D Three-dimensional structure of interaction between AFP protein and AFP_5, AFP_12 aptamers. PCT protein was presented in a surface form, displaying its structural features. Aptamers were depicted in a branched structure. Structures of all four aptamers binding to the PCT protein were predicted and visualized in the form of images (3D models on the left). Molecular interaction between PCT aptamers and the PCT protein was analyzed using Ligplot+. Hydrophobic interactions between aptamers (pink comb) and PCT (red comb) are shown. Hydrogen bonds are presented by green dots, with the length of each hydrogen bond indicated by a green number. Hydrogen bonds can increase the binding stability and affinity between aptamers and PCT

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Based on the results of structural analysis, how the four aptamers interacted with the PCT protein were determined. Each interaction predominantly involved hydrophobic interactions accompanied by hydrogen bonds. These hydrogen bonds formed at distances ranging from 1 to 4 Å. Through these findings, we were able to substantiate reasons behind the high binding affinity of the four aptamers that were selected based on their superior affinity in the previous experiments.

Electrophoretic mobility shift assay (EMSA) for confirmation of aptamer binding

To experimentally confirm binding between the PCT protein and aptamers, we performed EMSA, a traditional method used to assess interaction and binding between proteins and nucleic acids. Thus, we employed EMSA to experimentally determine the binding between PCT and aptamers.

Figure 5 presents result images of SDS-PAGE and Coomassie Brilliant Blue staining of PCT protein, confirming its size to be 17 kDa (Fig. 5, left). In EMSA, to maintain binding between PCT protein and aptamer, we used a native polyacrylamide gel without SDS detergent. The mixture of PCT protein and aptamer was subjected to electrophoresis. Nucleic acids were then visualized by staining with ethidium bromide (EtBr). Results showed that these aptamers could bind to PCT, which has a relatively larger molecular weight, appeared at the top of the gel, while unbound aptamers remained at the bottom. Figure 5 presents EMSA results and shows electrophoretic mobility shifts of the four selected PCT aptamers, indicating their binding to PCT protein in each lane after electrophoresis and EtBr staining. We confirmed that all four aptamers exhibited gel shifts due to their binding with PCT. In contrast, lane binding with a random pool that lacked specific affinity for PCT showed no gel shift, indicating no binding to PCT (Fig. 5, right).

Fig. 5
figure 5

Electrophoretic mobility shift assay (EMSA) for the validation of binding status. Before performing EMSA, the size and position of the PCT protein were confirmed using SDS-PAGE and Coomassie Brilliant Blue staining (image on the left). Subsequently, EMSA was conducted by incubating the four aptamers and a random pool of ssDNA with PCT. As a result, the four aptamers, which exhibited specific binding affinity for PCT, showed a visible signal of fluorescence intensity caused by EtBr at the position of the PCT protein. However, the random pool of ssDNA lacking specific binding affinity to PCT showed minimal signal at the same position

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Furthermore, by analyzing the intensity of the shifted bands, we can compare the binding affinities of each aptamer. PCT_Ap11 and PCT_Ap17 exhibited relatively intense bands compared to the other two aptamers, which is consistent with the results from previous experiments, indicating that these two aptamers have higher binding affinities.

Enzyme-linked immunosorbent assay (ELISA) for quantitative detection of PCT

We implemented an ELISA-based PCT detection system using PCT aptamers as probes. PCT antibodies were coated onto a 96-well plate. PCT proteins bound to the antibodies were detected using FAM-modified PCT aptamers (Fig. 6A). In the experiment, four aptamers (PCT_Ap1, 11, 14, and 17) with FAM were used as individual probes. All four aptamers were capable of detecting PCT. Among them, PCT_Ap11 and PCT_Ap17 aptamers exhibited the highest PCT detection signals (Fig. 6 B). To assess detection ranges of the two aptamers that displayed the highest signals, an experiment was conducted to observe changes in detection signals with varying PCT concentrations. Experimental results demonstrated that the fluorescent signal of FAM in both PCT_Ap11 and PCT_Ap17 aptamers was consistently increased as the PCT concentration increased (Fig. 6c and B). Each detection value was transformed into a logarithmic graph. The graph's slope (R-squared) was then calculated (PCT_Ap11: Y = 10,207*X + 46,285 (R-squared = 0.9914), PCT_Ap17: Y = 8308*X + 46,009 (R-squared = 0.9970)) (Fig. 6E). PCT_Ap17 exhibited a higher R-squared value, indicating a relatively higher level of result reliability. Both aptamers demonstrated R-squared values above 0.9900, indicating a high sensitivity in detecting PCT. This confirms that both aptamers have sufficient utility for PCT detection in the diagnosis of sepsis.

Fig. 6
figure 6

Detection sensitivity of the PCT platform based on aptamers. A Schematic diagram of the detection platform based on PCT aptamers for PCT detection under different test conditions. B Results of PCT detection assay performed according to the conditions described in (A). Among the four aptamers, PCT_Ap11 and PCT_Ap17 demonstrated relatively high PCT detection outputs. C PCT detection assay using PCT_Ap11 aptamer at varying PCT concentrations. D PCT detection assay using PCT_Ap17 aptamer at varying PCT concentrations. E Reliability of PCT detection assays using PCT_Ap11 and PCT_Ap17 (PCT_Ap11 represented in light blue and PCT_Ap17 in yellow). F Detection of PCT in serum. Both PCT_Ap11 and PCT_Ap17 aptamers demonstrated high ability to detect PCT present in serum

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These results indicate a high potential of both PCT_Ap11 and PCT_Ap17 aptamers for detecting PCT for early sepsis diagnosis, as validated by ELISA results. To further validate this, additional experiments were conducted to evaluate the specificity of PCT aptamers. For this purpose, PCT protein was spiked into a buffer as well as human serum containing various components. Detection specificity of the aptamer was then assessed. Experimental results for the specificity of PCT aptamers for PCT detection showed that both PCT_Ap11 and PCT_Ap17 aptamers were capable of specifically detecting PCT present in the serum (Fig. 6F). This confirmed that both PCT_Ap11 and PCT_Ap17 aptamers exhibited specificity for detecting PCT in human serum, even in the presence of other molecules. These results indicate that the PCT-aptamer-based PCT detection technology is capable of detecting PCT present in the blood or serum of actual patients, thereby enabling early diagnosis of sepsis. Its potential as a diagnostic tool for sepsis was demonstrated.

Sepsis is a disease that consistently causes deaths worldwide. It occurs when bacteria proliferate in the blood, resulting in production of toxic substances and various complications that can have a lethal impact on patients in a short period of time (Hotchkiss et al. 2016). Therefore, early diagnosis of sepsis is crucial. Currently, various methods are being used to diagnose sepsis. However, they have limitations in terms of detection limit and accuracy.

Discussion

Traditionally, there are several methods used for diagnosing sepsis, including ELISA, blood culture, white blood cell count, qPCR, and imaging techniques. Although blood culture has been the most traditional method, it has limitations in terms of accuracy and, notably, long detection time. This is a critical drawback in the diagnosis of sepsis, which requires prompt action. As an alternative, white blood cell count measurement can provide faster results. However, it has significantly reduced accuracy. qPCR, which amplifies bacterial genes in the blood, has a higher detection accuracy, up to 90%. Its drawback is its relatively longer detection time. On the other hand, imaging techniques such as X-ray or CT can be used. However, they require specialized equipment for imaging analysis, which is a disadvantage (Weinstein and Doern 2011; Vincent and Backer 2013; Gadsby et al. 2016; Sartori et al. 2011).

The most commonly used diagnostic method for sepsis, considering advantages and disadvantages of the aforementioned methods, is antibody-based ELISA. ELISA is widely employed. A wide range of target molecules are available for it. Representative markers utilized include CRP, Leptin, and IL-6. ELISA using these markers generally exhibits high sensitivity and fast detection time. However, these markers are also increased in general inflammatory conditions, not only in sepsis, which poses an inherent limitation in terms of detection accuracy. To overcome this limitation, PCT was utilized as a target molecule. PCT uniquely demonstrated specific utility in sepsis, distinguishing it from other targets. As a result, its diagnostic accuracy was significantly improved to levels ranging from 95 to 99% (Sager et al. 2017a; Pepys et al. 2012; El-Mashad et al. 2016; Franco et al. 2015) (Table 2).

Table 2 Traditional biomarkers for the diagnosis of bacteremia and diagnostic methods
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In this study, we developed a sepsis diagnostic platform utilizing PCT as the target molecule. The platform we developed was based on aptamers instead of antibodies, aiming to replace traditional antibody-based ELISA. This platform using PCT showed significantly enhanced detection sensitivity for sepsis diagnosis. The detection limit of PCT was < 0.5 ng mL–1 with antibody-based ELISA (Sager et al. 2017b). This falls short of the sensitivity of the aptamer-based PCT detection platform (0.001 ng mL–1) developed in this study.

The reason why our platform showed higher detection sensitivity than traditional antibody-based ELISA was due to different types of binding involved in antigen–antibody reaction and aptamer binding. All antigen–antibody bindings are physically weak interactions, including (I) Coulombic interactions, (II) Ca2+-bridges, and (III) Lifshitz–van der Waals interactions without a covalent bond (Oss et al. 1986). In contrast, aptamers could bind to their target molecules through hydrophobic interactions and hydrogen bonding, forming binding lengths within 4 Å with a strong binding. We confirmed these binding characteristics through in silico 3D structural analysis and conducted research to optimize the binding structure (Ghanty et al. 2022). This allowed us to develop an aptamer-based detection platform with optimal binding strength. This technique was also effectively utilized in our previous research (Oh et al. 2020; Sekhon et al. 2022; Shin et al. 2022; Lee et al. 2021). Therefore, in silico 3D structural analysis research holds great value in optimizing binding strength.

If we identify aptamer pairs that bind to different regions of the target molecule through in silico 3D structural analysis, it is indeed possible to construct a sandwich assay platform using only aptamers. This approach has also been extensively utilized in our previous research.

In this study, we used docking prediction as a computational method to predict the binding interactions between aptamers and PCT. While docking prediction provided valuable insights, its reliability as a predictive tool depends on experimental validation. Through various molecular biology methods and in silico validation techniques, we identified optimal aptamers with high affinity for PCT. These aptamers were incorporated into a sepsis diagnostic platform, which demonstrated excellent sensitivity and specificity for detecting PCT in both buffer and serum. The successful development of this aptamer-based platform represents a significant advancement in sepsis diagnosis and highlights the importance of combining computational predictions with experimental data for robust diagnostic technology development.

Results of this study confirmed that the aptamer-based PCT detection platform developed in this research has potential for early diagnosis of sepsis by detecting PCT in the body. Additionally, by incorporating a new molecule called aptamer and introducing in silico 3D structural analysis as a validation method during the platform development process, this study suggests widespread potential utilization of aptamers and in silico 3D structural analysis in future development of diagnostic technologies.