Heparin-Bead Extraction Enhanced Fluorogenic Aptamer–Thrombin Composite Reporter Enables Sensitive and Rapid Detection of Functional Antithrombin

Abstract

Antithrombin (AT) deficiency in the extracorporeal circulation during cardiac surgery leads to uncontrolled inflammation and vascular damage in patients. AT levels decrease in sepsis, major trauma, extracorporeal membrane oxygenation, and eclampsia. Monitoring plasma AT levels facilitates the accurate restoration of AT to baseline values through precise supplementation. Traditional methods of chromogenic assay and enzyme-linked immunosorbent assay (ELISA) kits encounter challenges, such as interference, inconsistency, and delayed response times, making real-time, reliable antithrombin monitoring a clinical gap. To address this critical need, we develop a heparin-bead extraction enhanced fluoroGenic aptamer–thrombin composite reporter (HExGATOR) for the rapid, sensitive, and precise detection of functional AT in plasma. Our design employs thrombin-binding aptamers and a fluorescence “turn on” technology such that a signal is produced upon the interaction of AT with the otherwise “turned off” aptamer–thrombin complex. The prominent signal-background interference originating from plasma is remarkably diminished by using a heparin-bead solid-phase extraction of AT. We achieved highly sensitive and rapid detection of AT in 5 to 20 min with a limit of detection of 15.11 nM. This approach offers a promising alternative to traditional AT tests in clinical settings, potentially facilitating personalized anticoagulant therapy.

Graphical Abstract

Antithrombin (AT), also termed antithrombin III (AT-III), is a plasma glycoprotein that inactivates several enzymes of the coagulation system, such as the serine protease human α-thrombin (factor IIa), and inhibits the blood clotting process involved in the coagulation cascade pathway.^1–3 The normal half-life of AT in plasma is approximately 3 days with a concentration of 2.3 μM.^4,5 AT interacts with thrombin, leading to the formation of AT–thrombin complexes that enable anticoagulation.^6,7 In this process, heparin augments the activity of AT by a conformational change and expedites its binding affinity to thrombin, thereby enhancing anticoagulation, whereas heparin alone has no direct anticoagulant effect.⁸

During cardiac surgery, blood bypasses the extracorporeal circulation to maintain blood fluidity in the circuit and prevent coagulation.^9,10 It also occurs to variable degrees in trauma, sepsis, eclampsia of pregnancy, and during extracorporeal membrane oxygenation (ECMO).^11–14 However, in circumstances when blood is exposed to the air or makes contact with other substances, coagulation happens along with the decrease of AT levels.^15,16 AT deficiency in plasma is usually followed by uncontrolled inflammation and vascular damage to patients, resulting in multiple organ failure and a high risk of mortality.^17–19 Heparin is usually the predominant agent used for coagulation treatment, particularly in the process of cardiopulmonary bypass (CPB).²⁰ However, the optimal dosage of heparin for administration and methods for monitoring its effects remain unresolved.^21,22 Currently, AT supplementation has become more popular since it improves patient survival.^23,24 However, excessive AT added into the plasma leads to a high potential for bleeding events.²⁵ To maintain AT at baseline levels, the functional AT (active with thrombin) concentration should be assessed first. To this end, a chromogenic-substrate-based commercial kit for the concentration measurement of functional AT has been developed. However, these commercially available assays perform unreliably in the presence of plasma, resulting from hydrolyzation of the chromogenic substrate with plasma.²⁶ Another approach to determine the concentration of AT is by using an ELISA-based kit; however, this strategy only detects the concentration of AT antigen using an AT-specific antibody, lacks functional information, and requires 4 h or more for measurement.²⁷ Therefore, a highly specific and fast platform to directly measure plasma functional AT concentration on site is urgently needed.

Herein, a heparin-bead-extraction enhanced fluoroGenic aptamer–thrombin composite reporter (HExGATOR) is developed for the sensitive and rapid detection of functional AT. Our reporter design employs thrombin-binding aptamers (TBA) coupled with a fluorescence “turn-on” technology to detect AT. Two distinct aptamers, TBA15 and TBA29, were selected for thrombin recognition. TBA15 is a 15-mer single-stranded DNA with sequence 5′-GGTTGGTGTGGTTGG-3′ and K_d ≈ 100 nM.²⁸ TBA15 binds to exosite I of thrombin, which is the binding site of fibrinogen. TBA29 is a single-stranded DNA with sequence 5′-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′ and K_d ≈ 0.5 nM.²⁹ It recognizes exosite II, which is involved in the activation of factors V/VIII. Modifications to the two aptamers were introduced to develop the fluorescence “turn-on” technology. The principle of this assay is shown below in Figure 1A. The 3′ end of TBA15 and the 5′ end of TBA29 are modified with polyethylene glycol (PEG) and then elongated with several bases (Table S1).³⁰ The elongated TBA15 is labeled with a FAM (fluorescein amidite) fluorophore at its 3′ end (TBA15-F), while the elongated TBA29 is modified with a Dabcyl (4-(dimethylaminoazo)benzene-4-carboxylic acid) quencher (TBA29-Q) at its 5′ end. In the presence of thrombin, TBA15 and TBA29 bind to exosite I and exosite II of thrombin, respectively, producing a dual aptamer–thrombin complex. The oligonucleotides at both ends of TBA15-F and TBA29-Q combine through nucleotide sequences installed to achieve proximity-dependent hybridization. Therefore, the distance between FAM and Dabcyl is reduced, and fluorescence from FAM is quenched by Dabcyl owing to fluorescence resonance energy transfer (FRET). When AT is added to this system, it combines strongly with thrombin, leading to the dissociation of the thrombin–aptamer complex (Figure 1A). Since the K_d for TBA15 is 100 nM, which is much smaller compared with that of TBA29, we hypothesize that the release of TBA15-F into the solution turns on fluorescence. To make the reporter capable of AT detection in intricate plasma samples, heparin Sepharose beads are used as solid-phase extraction (SPE) material to capture AT from human plasma due to the high affinity between heparin and AT, effectively eliminating interfering molecules that trigger background signals. The purified AT is then detected using a fluorogenic dual aptamer–thrombin reporter (Figure 1B). Since the reporter involves the direct interaction between AT and thrombin and the fluorescence recovery is proportional to AT concentration, this HExGATOR platform can be used for the quantitative detection of functional AT.

Figure 1.

Principle and workflow of the HExGATOR platform. (A) Principle of the aptamer–thrombin reporter. In the complex, two aptamers modified with a fluorophore and a quencher, respectively, bind to thrombin, and the fluorescence is quenched owing to FRET. In the presence of AT, the interaction between thrombin and AT leads to dissociation of the thrombin–aptamer complex, thereby turning on a fluorescence signal. (B) Workflow of HExGATOR. AT in human plasma is captured using heparin Sepharose beads to exclude interfering molecules. Then, purified AT reacts with the dual aptamer–thrombin complex.

METHODS

Reagents and Instruments.

DNA oligomers (Table S1) were purchased from Integrated DNA Technologies. Human antithrombin III was purchased from Grifols and Prolytix. Human alpha-thrombin (HCT-0020) was purchased from Prolytix. AT-III-deficient plasma (IATIIIDP1ML) was purchased from Innovative Research. Ultrapure water (DNase- and RNase-free) and 10× PBS solution were purchased from Invitrogen. Heparin Sepharose (ab193268) was purchased from Abcam, and 1 mL of beads was diluted with 4 mL of 1× PBS and used in the experiments. Heparin sodium salt was purchased from Santa Cruz Biotechnology. A BioTek SYNERGY H1 microplate reader was used to monitor fluorescence intensity during the reaction.

Aptamer-Based AT Detection Assay.

The aptamer–thrombin complex was prepared by incubating 200 nM μ-thrombin with 200 nM TBA15-F and TBA29-Q for 10 min at room temperature in PBS in a total volume of 90 μL per reaction. To detect AT, 10 μL of AT with various concentrations was added to the aptamer–thrombin complex solution. Real-time fluorescence signals generated from the reaction were monitored every 1 min by a microplate reader at 25 °C.

Heparin Bead-Based AT Extraction and Detection.

Heparin agarose beads were washed with PBS before extraction and then added to the samples to extract AT with agitation for 30 min in a thermal mixer at room temperature. After extraction, the beads were washed with PBS three times and the supernatants were discarded. Subsequently, heparin beads were mixed with the aptamer–thrombin complex (100 μL) for 20 min using an Eppendorf thermomixer at room temperature. Finally, the fluorescence intensities of the supernatants were tested using a microplate reader.

RESULTS AND DISCUSSION

Optimization and Characterization of the Aptamer-Based AT Detection Assay.

The aptamer–thrombin complex is pivotal for the detection of AT. The first step toward its formation is through the hybridization length region in both aptamers. A long length leads to aptamer hybridization, even without thrombin, resulting in analytical failures or reduced sensitivity. Conversely, a short complementary sequence may cause insufficient proximity-dependent hybridization and FRET. Aptamer sequences with a hybridization length varying from 7 to 10 nucleotides (nt) were tested to minimize spontaneous hybridization of the aptamers while intensifying proximity-dependent hybridization in the presence of thrombin (Table S1). In this experiment, 200 nM TBA15-F and TBA29-Q were mixed, followed by an analysis of the resultant fluorescence intensities, which were then normalized using the value from TBA15-F alone, as represented by (F + Q)/F, which reflects the degree of aptamer hybridization without thrombin. Figure 2A illustrates a decline in the normalized fluorescence intensity concomitant with extended hybridization sequence lengths, indicating that an increased proportion of TBA15-F and TBA29-Q underwent hybridization. We also assessed the fluorescence intensity upon the introduction of thrombin (200 nM) to initiate the formation of the aptamer–thrombin complex (F + Q + T), and a similar result was obtained (Figure 2A). The data suggested a hybridization length of 9 nt as it showed an optimal balance, maximizing aptamer–thrombin complex formation when compared to the 7 and 8 nt lengths while minimizing unwanted spontaneous hybridization between the aptamers in the absence of thrombin when compared to the 10 nt length.

Figure 2.

Formation of the aptamer–thrombin complex. (A) Optimization of aptamer hybridization length. F, Q, and T represent the fluorophore-modified aptamer (TBA15-F), the quencher-modified aptamer (TBA29-Q), and thrombin, respectively. (F + Q)/F is the fluorescence intensity ratio between F + Q and F only, and (F + Q + T)/F is the fluorescence intensity ratio between the complex F + Q + T and F only. The concentrations of aptamers and thrombin were 200 nM. (B) Optimization of aptamer concentration. Different concentrations of aptamers and thrombin with identical molar ratios were tested. (C) Buffer selection. Assay performance was tested in different buffers, including Tris-HCl buffer with K⁺ and Na⁺ (20 mM Tris-HCl, 5 mM KCl, 140 mM NaCl, pH 7.5), Tris-HCl buffer with K⁺, Na⁺, Mg²⁺, and Ca²⁺ (20 mM Tris-HCl, 5 mM KCl, 140 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, pH 7.5), PBS buffer, and PBS buffer with 1 mM MgCl₂ and 1 mM CaCl₂. All intensities were normalized by F. (D) Molar ratio optimization between aptamers and thrombin. Concentration was 200 nM each for the two aptamers. All intensities were normalized by F. (E) Comparison between FQT ratio 1:1:1 and 1:1:1.25 with the addition of AT. (F) Kinetics of aptamer–thrombin complex formation. All data were normalized by the fluorescence intensity at 0 min.

Next, the concentration of aptamers and thrombin was examined. Figure 2B depicts a decline in the (F + Q)/F value with increased aptamer concentrations, demonstrating an enhanced level of aptamer hybridization. The decline of (F + Q + T)/F was also observed in the presence of thrombin. Both 200 and 300 nM concentrations presented the most pronounced signal difference between (F + Q)/F and (F + Q + T)/F, so 200 nM was selected for the subsequent preparation of aptamer–thrombin complex.

Subsequently, the impact of various buffers on the aptamer–thrombin complex formation was investigated. As shown in Figure 2C, while the complex demonstrated similar fluorescence signals across four buffers, the spontaneous hybridization of the aptamers in the absence of thrombin differed remarkably. In particular, PBS buffer supplemented with Mg²⁺ and Ca²⁺ induced the most notable hybridization of the two aptamers, indicating the significant impact of bivalent ions on spontaneous hybridization compared with monovalent ions. In contrast, minimal spontaneous hybridization was observed with PBS alone and Tris-HCl buffer (K⁺ and Na⁺); however, the formation of the aptamer–thrombin complex (F + Q + T) in a Tris-HCl (K⁺ and Na⁺) environment also weakened, leading to the decision to use PBS for subsequent experiments. The optimal molar ratio between aptamers and thrombin for the formation of the complex was interrogated. In this test, a consistent concentration of the two aptamers (200 nM) was maintained while varying thrombin levels. As Figure 2D demonstrates, the fluorescence intensity stabilizes when the ratio of thrombin to aptamers exceeds 1:1, signifying complete formation of the complex. To determine the most suitable concentration for AT detection, AT was introduced into the complex, prompting the release of the aptamers. Figure 2E reveals that a complex with an equal proportion of aptamers and thrombin (1:1:1) yielded a higher fluorescence signal after incubation with AT, establishing 1:1:1 as the ideal ratio for preparing the aptamer–thrombin complex. Excess thrombin present in a solution with a 1:1:1.25 ratio could occupy the AT, thereby weakening the effective interaction between the complex and AT to produce the fluorescence signal.

Finally, the kinetics of aptamer–thrombin complex formation was investigated. The reduction in fluorescence intensity signals the formation of the aptamer–thrombin complex (Figure 2F). This process achieved equilibrium after a 10 min incubation period, resulting in a corresponding fluorescence intensity decrease of 66.45%.

Following the optimization of aptamer–thrombin complex formation, this assay was employed in the detection of AT. Figure 3A illustrates the real-time monitoring of the assay testing varying concentrations of AT ranging from 2 μM to 20 nM. Results indicated that the signal intensity was proportional to the AT concentration and that faster kinetics was obtained when a higher AT concentration was applied. Furthermore, the analytical performance of this assay was examined by analyzing the end-point signals corresponding to AT concentration at the 20 min mark (Figure 3B). The limit of detection (LoD) was determined to be 42.51 nM (3σ) with a strong linear relationship between signal and target concentration from 50 nM to 2 μM (R² = 0.9870), highlighting the assay’s sensitivity, robustness, and rapidness in AT detection.

Figure 3.

Analytical performance of the aptamer-based AT detection assay. (A) Real-time fluorescence signal monitoring of the aptamer-based assay for the detection of AT-III; the reaction volume was 100 μL. (B) Linear calibration of the assay. The reaction time was 20 min, and error bars indicate one SD (n = 5).

Heparin Bead-Based Extraction of AT in Human Plasma.

The aptamer–thrombin fluorogenic reporter was subsequently applied to detect AT in human plasma. However, an abnormally high fluorescence signal was observed when directly adding plasma to the assay wherein the signal intensity derived from 10× diluted human plasma (the approximate AT concentration is 230 nM) paralleled the signal from 2 μM pure AT. The undesired signal elevation persisted, even upon an 80-fold dilution of the plasma (Figure S1). This phenomenon indicates the presence of interfering molecules in plasma that trigger aptamer dissociation, creating nonspecific signals. For example, fibrinogen also interacts with the TBA15 binding site, exosite I, of thrombin and is converted to fibrin by thrombin to form clots. The competitive binding of fibrinogen to exosite I probably leads to the fluorescence leakage from the dissociation of TBA15-F, resulting in nonspecific signals.³⁰ This result emphasizes the necessity of an alternative approach. Given the affinity of heparin for AT and its role in enhancing AT activity as outlined in the Introduction,⁸ our approach leverages this interaction to augment fluorescence signal output by rapidly extracting AT from plasma.

To validate the hypothesis of heparin-based extraction, the effect of heparin on the aptamer–thrombin complex-based assay was tested first. Figure 4A depicts the fluorescence signal when heparin is added to the reaction. Specifically, the rapid rise in signal indicates heparin’s positive effect on expediting the binding between AT and thrombin, yet the excessive presence of heparin has no significant effect on the signal. Additionally, a comparison of the signal intensity under various concentrations of AT with and without heparin was conducted (Figure 4B), revealing a consistent acceleration of our aptamer assay by heparin. These findings indicate that heparin improves the reaction kinetics, consistent with the discovery that heparin enhances AT activity and increases its binding affinity to thrombin due to conformational changes in AT.⁸

Figure 4.

Heparin bead-based extraction of AT in plasma. (A) Influence of heparin on the aptamer-based AT detection assay. Real-time signals were generated with various amounts of heparin from 500 nM to 2 μM. The AT concentration is 500 nM. (B) Comparison of real-time signals from representative AT concentrations (100 nM, 500 nM, 1 μM) without or with heparin (2 μM). (C) Feasibility test of heparin beads. dP and B represent deficient plasma and heparin beads, respectively. NC w/B is the NC of the aptamer assay in the presence of beads. dP + AT + B represents the detection of AT spiked in dP and then extracted by beads. PC is the positive control of the aptamer assay, detecting 2 μM pure AT. (D) Optimization of heparin bead volume. Extraction volume was 100 μL with 2 μM AT in this experiment. (E) Optimization of extraction time. 30 μL of beads was incubated with 2 μM AT (100 μL). (F) Optimization of extraction volume. 30 μL of beads was incubated with AT for 30 min, and the amount of AT spiked was kept the same in different extraction volumes. All experiments were conducted in triplicate and normalized by the PC (detection of the same amount of AT without extraction).

By analyzing the signals corresponding to AT concentration at the 5 min mark, a LoD of 55.61 nM (3σ) was obtained (Figure S2). Adding heparin shortens the assay time from 20 to 5 min while maintaining analytical performance similar to the nonheparin assay.

Next, heparin Sepharose beads were explored for their effectiveness as a SPE material for the purification of AT from plasma samples. AT-deficient plasma was utilized to avoid the impact of endogenous AT on our assay. The protocol involves incubating these beads with plasma, followed by supernatant removal, washing, and subsequent combination with the aptamer–thrombin complex assay for AT detection (Figure 1). As shown in Figure 4C (column dP), AT-deficient plasma triggers fluorescence, reaffirming that plasma contains species capable of inducing nonspecific signals. Remarkably, after incubating AT-deficient plasma with the SPE material, these beads led to a substantial signal reduction (Figure 4C, column dP + B) that aligned closely with the bead-based negative control (NC) (Figure 4C, column NC w/B), demonstrating that the extraction step eliminates the nonspecific signal dramatically. It is noteworthy that heparin can bind a variety of proteins,³¹ so the extraction step might not be exclusive to AT. However, after extraction, a signal similar to that of the NC implies that other heparin-binding proteins are less likely to trigger the aptamer–thrombin complex assay. Furthermore, the beads could isolate 2 μM AT purposefully spiked in AT-deficient plasma (Figure 4C, column dP + AT + B), yielding a signal level of 96.7% of the positive control (PC) (Figure 4C, column PC). This indicates that the interaction between the aptamer–thrombin reporter and the immobilized (not free in solution) AT does not compromise signal intensity, demonstrating the feasibility of the heparin bead-based method.

Fine-tuning of the extraction parameters commenced with altering bead volume. Figure 4D reveals that a volume of 30 μL of beads is optimal as it outperformed 10 and 20 μL in signal intensity and was similar to that of 40 μL. Next, extraction time and volume were optimized. As depicted in Figure 4E, a 30 min incubation time emerged as the most effective. An extraction volume of up to 500 μL without a notable compromise of extraction efficiency was observed, and 100 μL was selected for the following tests (Figure 4F).

HExGATOR Platform for Detection of Functional AT in Human Plasma.

Analytical performance of the HExGATOR platform was interrogated next. As depicted in Figure 5A, the LoD of our method for the detection of pure AT is 30.53 nM (3σ) with a linear range from 50 nM to 2 μM (R² = 0.9814). To further evaluate the precision of the method, interassay and intra-assay tests were conducted. The results indicate that the mean relative standard deviation (RSD) for the intra-assay test ranges between 0.34 and 2.67%, while the RSD for the interassay test is 2.59% (Figure 5B). These findings confirm the high repeatability and reproducibility of the method. In addition, extractions using AT-deficient plasma spiked with varying concentrations of AT were conducted. The spiked samples exhibited a dose-dependent increase in fluorescence signal intensity with a LoD = 15.11 nM and a linear range from 20 nM to 2 μM (R² = 0.9912), suggesting that the method reliably detects different levels of AT in human plasma (Figure 5C). Figure 5D indicates that the assay can identify AT in spiked plasma samples with an excellent recovery rate (102.4–112.4%) and RSD (1.32–6.90%). The HExGATOR platform outperforms the original assay (Figure 3) in sensitivity (15.11 nM vs 42.51 nM) and offers a wider linear range (20 nM–2 μM vs 50 nM–2 μM), indicating the superior analytical performance of our strategy. It is worth noting that AT deficiency is defined as having an AT level below 70%, ranging from 40 to 70% (0.92–1.61 μM).^32,33 Considering the pathological level of AT, the HExGATOR platform is sensitive enough to meet the requirements of clinical diagnostics.

Figure 5.

Analytical performance of HExGATOR for the detection of AT. (A) Calibration curve of the HExGATOR platform for the detection of AT in the standard solution with various concentrations (20 nM–2 μM; the final volume was 100 μL). The reaction time is 20 min. Error bars indicate one SD (n = 3). (B) Intra-assay and interassay RSD of the HExGATOR platform. The AT concentration is 500 nM. (C) Calibration curve of the HExGATOR platform for the detection of AT in plasma samples. Plasma (AT-deficient, 20 μL) is diluted 5-fold in PBS and spiked with various AT concentrations (20 nM−2 μM, final volume was 100 μL). The reaction time is 20 min. Error bars indicate one SD (n = 3). (D) Recovery study of various concentrations of AT detected in spiked plasma samples.

CONCLUSIONS

Today, therapeutic intervention with AT is limited because clinicians have no way to detect how the functional levels are being consumed and, consequently, no way to judge the efficacy of therapy. AT therapy could be more broadly studied in CPB, ECMO, eclampsia, trauma, and sepsis if a reliable bedside test for functional AT levels was available. Herein, we have successfully developed an innovative, sensitive, and rapid platform called the HExGATOR for the detection of functional AT in human plasma. By leveraging the specific interaction between AT and thrombin and employing the fluorescence “turn on” strategy based on TBA, this assay effectively signals the presence of AT in plasma with a 15.11 nM sensitivity and a dynamic range from 20 nM to 2 μM. Through optimization studies, the robust formation of the aptamer–thrombin composite reporter essential for the assay’s functionality was accomplished. Moreover, the challenge of plasma off-target protein interference was solved by introducing a heparin bead-based extraction strategy, effectively purifying AT from human plasma samples and significantly enhancing the assay’s sensitivity, specificity, and accuracy in a real biological matrix.

Our method holds several advantages, including high sensitivity and specificity, quick analysis time, and simplicity. Compared to other commercially available AT detection kits, our method determines the functional AT level rather than AT antigen concentration, demonstrating superior performance in both assay time and precision while maintaining the sensitivity required for clinical diagnostics (Table S2). These features underscore its potential application in clinical settings, especially in scenarios requiring immediate anticoagulation status assessment. Furthermore, our approach offers a promising alternative to traditional coagulation tests, potentially facilitating personalized anticoagulant therapy by providing precise, real-time insights into patients’ antithrombin levels. Additionally, the simplicity of our assay also provides the potential for automation and miniaturization, paving the way for a point-of-care (POC) testing device, thus broadening the accessibility and applicability of this technology in various healthcare settings and advancing medicine by allowing for new knowledge and advanced therapeutic interventions.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.4c01294.

Additional information and results including direct detection and linear calibration using the aptamer-based assay, DNA sequences, and comparison of this method with other commercial AT detection kits (PDF)