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. 2021 Oct 26;7(11):1898–1907. doi: 10.1021/acscentsci.1c00948

Clinical Trial: Magnetoplasmonic ELISA for Urine-based Active Tuberculosis Detection and Anti-Tuberculosis Therapy Monitoring

Jeonghyo Kim †, Van Tan Tran †,‡, Sangjin Oh †, Minji Jang §, Dong Kun Lee ∥, Jong Chul Hong ⊥, Tae Jung Park #, Hwa-Jung Kim ∇,*, Jaebeom Lee †,○,*
PMCID: PMC8614099  PMID: 34841060

Abstract

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The coronavirus disease 2019 (COVID-19) pandemic has proved the importance of fast and widespread diagnostic testing to prevent serious epidemics timely. The first-line weapon against rapidly transmitted disease is a quick and massive screening test to isolate patients immediately, preventing dissemination. Here, we described magnetoplasmonic nanozymes (MagPlas NZs), i.e., hierarchically coassembled Fe3O4–Au superparticles, that are capable of integrating magnetic enrichment and catalytic amplification, thereby the assay can be streamlined amenable to high-throughput operation and achieve ultrahigh sensitivity. Combining this advantage with conventional enzyme-linked immunosorbent assay (ELISA), we propose a MagPlas ELISA for urine-based tuberculosis (TB) diagnosis and anti-TB therapy monitoring, which enables fast (<3 h), and highly sensitive (up to pM with naked-eyes, < 10 fM with plate reader) urinary TB antigen detection. A clinical study with a total of 297 urine samples showed robust sensitivity for pulmonary tuberculosis (85.0%) and extra-pulmonary tuberculosis (52.8%) patients with high specificity (96.7% and 96.9%). Furthermore, this methodology offers a great promise of noninvasive therapeutic response monitoring, which is impracticable in the gold-standard culture method. The MagPlas ELISA showed high sensitivity comparable to the PCR assay while retaining a simple and cheap ELISA concept, thus it could be a promising point-of-care test for TB epidemic control and possibly applied to other acute infections.

Short abstract

Multifunctional Fe3O4−Au nanozyme-integrated ELISA enables fast and highly sensitive urinary tuberculosis biomarker detection and noninvasive therapeutic response monitoring in patients’ urine.

Introduction

Historically, infectious diseases were one of the greatest threats and continue to cause catastrophic damage globally. The coronavirus disease 2019 (COVID-19) pandemic hit, devastating global society and the economy. SARS-CoV-2 is highly contagious and rapidly spreading across the world. One of its mysteries is the emergence of asymptomatic infections that contribute to the silent spread of the virus, causing serious difficulties in epidemic control.1 An ancient human disease, tuberculosis (TB), still causes millions of deaths annually, despite the availability of effective treatment due to the lack of rapid and accurate diagnostic tests. More seriously, about one-quarter of the world’s population has latent TB, which potentially can be reactivated, resulting in transmitting the bacteria to others.2 Thus, one thing is clearly highlighted amid the current global health crisis: the most effective way of containing the rapid and silent spread of epidemics is a quick and massive screening test to isolate patients as early as possible, preventing dissemination and initiating faster patient care.3,4

Several TB diagnostic tests have newly been developed, but still many constraints exist toward point-of-care (POC) application. Isolation of Mycobacterium tuberculosis (Mtb) by a culture system (Bactec MGIT 960, BD Diagnostics and BacT/Alert MB, bioMerieux) remains the most accurate and the gold standard, but the culture takes weeks and is not accessible to resource-constrained regions.5 The polymerase chain reaction (PCR)-based GeneXpert MTB/rifampin (RIF) assay (Cepheid) is the most advanced tool, but this shows poor performance in low bacterial loads, such as extrapulmonary TB (EPTB), and is not affordable to resource-limited settings.6 The IFN-γ release assays (IGRAs) have received negative policy recommendations from World Health Organization (WHO) due to the inability to distinguish active TB and latent TB infection (LTBI).7 The lateral flow urine lipoarabinomannan (LF-LAM) assay (Abbott Diagnostics) is a potential TB POC test but has shown suboptimal sensitivity;8 WHO only recommended this test to assist TB in HIV-positive diagnosis.5,9 Thus, there is an urgent need for an accurate and rapid POC test to decrease death rates and reduce TB transmission.

The multifunctional nanozymes (NZs), which are enzyme-mimic nanocomposites with various multifunctional combinations, offer an intriguing strategy for high-performance biomedical systems.10 In the past several years, highly diversified multifunctional NZs have been introduced for biosensing,10,11 photothermal therapy,12,13 fluorescence imaging,14 magnetic resonance imaging (MRI),15,16 antioxidants,17 and magnetic separation/recycling.18 In previous reports, we have developed magnetic/plasmonic nanoparticle (NP)-based sensing platforms for TB and influenza monitoring such as the plasmon-enhanced fluoro-immunoassay,19,20 magnetophoretic assay,21,22 and NZ-linked immunosorbent assay (NLISA).23−27 In particular, the combination of NZ and magnetic enrichment has been a fascinating strategy to achieve high sensitivity. In this context, we designed a new type of multifunctional NZs by coassembling Fe3O4–Au superparticles, i.e., magnetoplasmonic (MagPlas) NZs, to acquire both magnetic and catalytic enhancement in a single biocatalyst. Assembling of the plasmonic Au on the Fe3O4 core could provide (i) dramatically enhanced catalytic activity of Fe3O4 NPs, which are considered as having relatively low activity,28−30 and (ii) an easy and highly efficient conjugation route for various bioreceptors, including thiol-terminated peptides or metal-binding peptides.21,22,27

Here, we report a clinical trial using MagPlas NZs with the conventional enzyme-linked immunosorbent assay (ELISA), called MagPlas ELISA, which is proposed for urine-based active TB detection and anti-TB therapy monitoring. In particular, we aimed to develop a nanotechnology-based “End-TB” technique that circumvents current limitations in TB epidemic control tools and is applicable to use in clinical practice. The MagPlas ELISA performs simultaneous magnetic enrichment and catalytic amplification using a Fe3O4–Au NZ reagent in clinical urine, after which the color generation confirms the detection of the target antigens. This strategy has several practical advantages: (i) Target antigens (CFP-10 and Ag85, both are the earliest secreted antigens in large amounts)22,31,32 can be directly enriched from a patient’s urine. Urine seems to be ideal for POC testing because sampling is easy for both adults and children, noninvasive, of large quantity, and poses no infection risk.5 (ii) Antigen detection is straightforward evidence of Mtb growth, thus it can discern active TB from nontuberculous mycobacteria (NTM) infection and LTBI, which failed in Bactec MGIT and IGRAs.7,21,22 (iii) Noninvasive EPTB diagnosis and treatment monitoring are significant. Diagnosis of EPTB is challenging due to the paucibacillary nature of Mtb, difficulties in accessing infected sites, and variable symptoms similar to other diseases,33 hence it poorly performs in Bactec MGIT and Cepheid GeneXpert. (iv) The reduced cost (∼US $3 per test) and rapid clinical decision (<3 h) can be useful for POC TB diagnostics in resource-limited countries.

Results and Discussion

MagPlas ELISA for Mtb Infection Diagnoses

The dual-functional MagPlas NZs are a promising replacement of natural peroxidase/catalase for the conventional in vitro diagnostic (IVD) tests such as ELISA, or the lateral flow assay (LFA). The MagPlas NZs are superstructured Fe3O4–Au NPs. First, 10 nm Fe3O4 nanocrystals construct hundreds of nanometer clusters, then hierarchically coassemble with plasmonic Au particles, which could achieve (i) superparamagnetism with a sufficient saturation magnetization of 63.2 emu/g, both essential for rapid and repeated magnetic separation,34 and (ii) enhanced peroxidase-like activity (>6.5- and >60-times higher than that of Fe3O4 NPs and horseradish peroxidase, respectively) caused by the synergistic effect of the Fe-plasmonic system that intensifies the chromogenic coloration28−30 (further details on the physical and catalytic properties of NZs are given in Supporting Information 1–3).

Figure 1 illustrates the MagPlas ELISA, designed for urine-based active TB diagnosis. Several Mtb-secretory antigens, such as ESAT-6, CFP-10, Ag85, and MPT64, can be found in patient urine after being filtered from the kidney (protein molecular weight <67 kDa).32,35−37 We selected CFP-10 and Ag85 as the target biomarkers because they are abundantly secreted in the early stage of the disease and are direct evidence of virulent Mtb growth, hence a strong indicator of active Mtb infection.22,31,32 To demonstrate the multiplexed POC testing, two types of kits were fabricated, i.e., the TB CFP-10 kit and TB Ag85 kit, by labeling with different pairs of antibodies, and thus they are capable of performing parallel analysis of multiple markers relevant to TB infection (Figure S14). Moreover, in this study, we have employed genetically engineered recombinant antibodies (GBP-G2 and SBP-G3 for CFP-10 and GBP-50B14 and SBP-8B3 for Ag85, respectively), in which the gold binding protein (GBP) or silica binding protein (SBP) was fused to the N-terminus of the Fab fragment of antibodies (G2, G3, 50B14, or 8B3; Figure S14b). Since the GBP/SBP regions have a strong and specific affinity for the Au/SiO2 surfaces,38 this offers advantages not only for highly oriented immobilization but also facile protein conjugation.36,39 The MagPlas ELISA kit is composed of the NZ–antibody conjugates and a 96-detection-well plate (Figure S15). Briefly, the NZs were directly conjugated with GBP-Fab antibodies through the GBP-Au (111) lattices’ specific affinity. While for the counterpart, a thin layer of polydimethylsiloxane (PDMS) was cured on the surface of each well, followed by immobilizing the SBP-Fab antibody through SBP-SiO2 adsorption.

Figure 1.

Figure 1

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Magnetoplasmonic ELISA for Mtb infection diagnoses. Schematics showing the overview of the procedural steps based on the “3C immunoassay strategy”. The MagPlas NZs were functionalized with GBP-antibody that was used as a magnetic separator and a signal amplifier. Step 1: Mtb-specific biomarkers are directly captured by antibody-labeled MagPlas NZs in a urine sample (Capture) and then magnetically collected, which enriches targets (Concentration). Step 2: The target captured MagPlas NZs were transferred on the antibody conjugated 96-well plate, then conjugated in a sandwich structure. A catalytic reaction was initiated by the addition of substrate reagents, and finally, an amplified color signal is generated (Coloration).

The assay directly analyzes the patient’s urine, which starts by mixing clinical samples with NZs. During the incubation, the target proteins are captured and extracted through immunomagnetic enrichment. The target captured-NZs are sequentially transferred to the detection plate, resulting in binding with a complementary antibody. Finally, the chromogenic substrate (3,3′,5,5′-tetramethylbenzidine, TMB) and hydrogen peroxide (H2O2) are added to each well of the plate; in the presence of the analyte, the NZs are immunologically bound on the surface of the well plate and act to disproportionate H2O2, allowing the oxidation of TMB. As a consequence, the oxidized TMB generates a distinct blue-colored solution (λmax = 652 nm, ε652 nm = 39,000 M–1 cm–1), then finally turns to yellow (i.e., diimine, λmax = 450 nm) after terminating the catalytic reaction. The absorbance measured at 450 nm (Abs@450 nm) is proportional to the amount of NZs on the well plate, and thus the concentration of the captured biomarkers, which enables quantification of target proteins.

Sensitive and Selective Detection of TB Biomarkers with MagPlas ELISA

Figure 2a and b show the results for the detection of CFP-10 and Ag85 with various concentrations. The yellow-colored signals (λmax = 450 nm) were read out using a conventional plate reader. A logarithmically linear regression curve between the Abs@450 nm and antigen concentration was obtained for both kit types. The linear range was observed from femtomolarity to micromolarity with a correlation coefficient of R2 > 0.96. The limit of detection (LOD) was calculated to be 2.5 fM and 1.6 fM for the TB CFP-10 kit and TB Ag85 kit, respectively. This ultralow LOD is attributed to the duplicated signal enhancement mechanism and concomitant increases in naked-eye detection sensitivity up to the picomolar level.

Figure 2.

Figure 2

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Detection sensitivity and selectivity of the multiplexed MagPlas ELISA kits. Calibration curves of (a) the TB CFP-10 kit and (b) the TB Ag85 kit (n = 3; R2 > 0.96). Serially diluted CFP-10 and Ag85 samples were applied for each kit, and the dynamic range was shown in the range from femtomolar to micromolar. (c,d) The selectivity tests of the multiplexed MagPlas ELISA kits. Nine tests were run with different combinations of the TB antigens: (i) phosphate-buffered saline (PBS, pH 7.4), (ii) non-TB control urine, (iii) CFP-10, (iv) Ag85, (v) HspX-16, (vi–viii) combinations of two antigens, and (ix) all three antigens (the concentrations of the mixed antigens are kept constant for all combinations: CFP-10 8.3 nM, Ag85 3.9 nM, and HspX-16 112.5 nM). The correlated colorimetric signals were visualized by the color map in a, b, and d.

The selectivity of the assay is intimately related to the exquisite specificity of the antibodies and the sensor design that minimizes the nonspecific reaction. To validate the selectivity of the MagPlas ELISA, nine sets of assays were performed with different combinations of the TB antigens (Figure 2c,d, see the legend for further detail). CFP-10 and Ag85 are target antigens for TB CFP-10 kit and TB Ag85 kit, respectively. The nontargeted antigen, heat shock protein X-16 kDa (HspX-16), and their mixtures with targeting antigens were tested to rule out feasible cross-reactivity of G2, G3, 50B14, and 8B3 antibodies prior to clinical assessment because the HspX is known as a cytoplasmic Mtb antigen secreted in early stages.21,27 The concentrations of the mixed antigens are kept constant for all combinations: CFP-10 (8.3 nM), Ag85 (3.9 nM), and HspX-16 (112.5 nM). As shown in Figure 2c and d, a positive signal (yellow color) is accurately correlated with the presence of the target antigen in each combination, which is clearly distinguishable from the background signal in both kits. The spiked unrelated antigens in the mixed solution are not recognized by antibodies and hence not contributed to the color generation. Therefore, these results support the non-cross-reactivity of antibodies and the minimized nonspecific reaction in the assay.

Diagnosis of Active Pulmonary Tuberculosis (PTB) with MagPlas ELISA

The straightforward detection of urinary antigens with an ELISA format could be an attractive alternative to cumbersome standard methods. However, to implement the MagPlas ELISA for practical patient care, it is essential to demonstrate the effectiveness of the assay against possible interferences found in real samples. A validation study was designed with 297 clinical urine samples: pulmonary tuberculosis (PTB) patient (n = 100), non-TB control group (n = 122), extra-pulmonary tuberculosis (EPTB) patients (n = 36), and non-EPTB patients (lymphadenitis and lymphoma, n = 39; Figures 3 and 4).

Figure 3.

Figure 3

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Diagnosis of active pulmonary tuberculosis (PTB) with MagPlas ELISA. (a) Detection of urinary CFP-10 and Ag85 antigen levels in PTB groups: a cohort of AFB-positive patients (n = 90), AFB-negative patients (n = 10), and a non-TB control group (n = 122). (b) Color maps of obtained Abs@450 nm intensities in a. Each column represents the result from the TB CFP-10 kit (upper cell) and TB Ag85 kit (lower cell). (c) A ROC curve analysis. (d) Box plots for CFP-10 and Ag85 levels for the three groups. Data are expressed as mean ± s.d. of triplicate measurements. In the box plots in d, data points with median (center line), 1%, 25%, 75%, and 99% value lines (box lines) are presented. Significance is defined as *P < 0.05.

Figure 4.

Figure 4

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Noninvasive diagnosis of extrapulmonary tuberculosis (EPTB) and treatment monitoring with MagPlas ELISA. (a) Detection of urinary CFP-10 and Ag85 antigen levels in EPTB groups: a cohort of TB lymphadenitis (n = 36) and non-EPTB patients (n = 39), which is composed of two subgroups: lymphadenitis (n = 25), and lymphoma (n = 14). (b) Colormap of obtained Abs@450 nm intensities in a. Each column represents the result from TB CFP-10 kit (upper cell) and TB Ag85 kit (lower cell). (c) A ROC curve analysis. (d) Box plots for CFP-10 and Ag85 levels for the EPTB patient groups. The non-TB control data involved in c and d are identical to the result shown in Figure 3. (e) Longitudinal monitoring of urinary CFP-10 and Ag85 in TBLN patients’ sample during therapeutic treatment. Data represent the average and the s.d. of triplicate measurements. In the box plots in d, data points with median (center line), 1%, 25%, 75%, and 99% value lines (box lines) are presented. Significance is defined as *P < 0.05.

Figure 3 shows the results of PTB diagnostic testing. One-hundred urine samples were obtained from PTB patients whose Mtb infections were determined by the standard bacteriological methods and PCR tests with their sputum (Table S3). Among these, 10 samples showed a smear and culture negativity (acid-fast bacillus (AFB) negative) and were only positive in the PCR test, which, mainly due to the fewer bacilli in the specimen and, concomitantly, a lower positive rate, is expected in standard tests. And 90 samples showed smear and/or culture positivity (AFB positive). Non-TB control samples (n = 122) proven by bacteriology and chest X-rays were included to exclude the possibility of false-positive results. Overall, the assays with PTB samples showed higher signals regardless of the antigen type (Figure 3a, P < 0.05). Remarkably, the MagPlas ELISA was able to distinguish distinctly PTB samples by their yellow color signal, whereas the samples from the non-TB control group yielded a transparent or a lightly colored solution (Figure 3b). Furthermore, the signal readout with a conventional plate reader allows an accurate quantification of the concentration of the target antigen. Next, receiver–operation characteristic (ROC) curve analyses were performed using CFP-10 and Ag85 levels as a predictor (Figure 3c). The area under the ROC curve (AUC) value of the TB CFP-10 kit was 0.92, whereas the TB Ag85 kit yielded a lower AUC of 0.63. The optimal cutoff values (Abs@450 nm) that maximize both sensitivity and specificity, i.e., the points on the ROC curves with a maximum Youden’s index, were 0.207 and 0.255 for TB CFP-10 kit and TB Ag85 kit, respectively. With these cutoffs, the TB CFP-10 kit diagnosed 85 of 100 (85.0%) PTB cases correctly, with 85.6% sensitivity in AFB-positive cases and 80.0% sensitivity in AFB-negative cases. The TB Ag85 kit can diagnose PTB accurately in only 47 of 100 (47.0%) cases, with 51.1% and 10.0% sensitivity in AFB-positive and -negative cases, respectively. All the non-TB control samples yielded a signal under the cutoff levels, and the specificities of the TB CFP-10/Ag85 kits were 96.7% and 92.6%, respectively (Figure 3d, Table 1). Thus, far, the MagPlas ELISA urine test showed a robust diagnosis in PTB patients, particularly in AFB-negative cases with sensitivity exceeding current conventional methods.

Table 1. Sensitivity and Specificity of MagPlas ELISA Kits for PTB Diagnosis.

  cutoff valuea (Abs@450 nm) AUC group (n = 222) positive results/total no. sensitivity, % specificity, %
TB CFP-10 kit 0.207 0.92 pulmonary TB (n = 100)   85.0  
AFB-positive 77/90 85.6  
AFB-negative 8/10 80.0  
non-TB (n = 122) 4/122   96.7
TB Ag85 kit 0.255 0.63 pulmonary TB (n = 100)   47.0  
AFB-positive 46/90 51.1  
AFB-negative 1/10 10.0  
non-TB (n = 122) 9/122   92.6
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a

Cutoff values of both kits were established on the maximum Youden index value in a tested patient’s groups.

Noninvasive Diagnosis of Extrapulmonary Tuberculosis (EPTB) and Treatment Monitoring with MagPlas ELISA

Tuberculous lymphadenitis (TBLN), which accounts for the largest proportion of extrapulmonary TB (EPTB), should be suspected in a patient with swollen lymph nodes that are firm, asymmetric, and larger than 2 cm in diameter. However, this symptom is also common for various causes, such as HIV-related lymphadenopathy, malignancies, and other lymph node infections, thus it is difficult to differentiate them clinically.40 A fine-needle aspiration (FNA) of the lymph node with biopsy is highly suggested to rule out the possibility of lymphoma (cancer of the lymphatic system), because that is the only assured way of determining whether a grown node is malignant or benign. Therefore, if a quick and reliable TBLN discrimination from other lymphadenopathies is possible in a parallel screening test, that would be a breakthrough to aid clinicians’ decision making in a variety of clinical scenarios.

A total of 75 urine samples from TBLN patients (n = 36) as well as non-TBLN patients (n = 25 for lymphadenitis and n = 14 for lymphoma) were collected from the Department of Otorhinolaryngology, Dong-A University Hospital for the past 5 years. Among them, nine TBLN patients’ health status was followed using a MagPlas ELISA for 26 weeks after the first hospital visit. All 75 patients showed symptoms of enlarged cervical lymph nodes and underwent the FNA with histological examination and a PCR test to confirm disease status (Tables S4). Figure 4 shows the performance of the MagPlas ELISA for noninvasive TBLN diagnosis and therapy monitoring. We observed higher levels of urinary CFP-10 in TBLN patient samples than those in non-EPTB control group (P < 0.05), which can be intuitively perceived with the naked eye as shown in the color map data. In contrast, the Ag85 antigens were undetectable or detected only weakly in TBLN urine samples, thereby they could not conclusively differentiate between TBLN and non-EPTB groups (Figure 4a, b, and d). From the ROC curve analyses, the AUC for the TB CFP-10 kit was 0.76, and the optimal cutoff value (Abs@450 nm) was determined as 0.248 (Figure 4c). With this cutoff value, the CFP-10 test was able to differentiate 52.8% (19/36) of TBLN cases from the control groups with a high specificity of 96.9% (155/161). However, the TB Ag85 kit yielded a poor AUC value of 0.49; consequently, it showed an unsatisfactory level of sensitivity (30.6%) and specificity (81.4%) even with an optimal cutoff value of 0.212 (Table 2).

Table 2. Sensitivity and Specificity of MagPlas ELISA Kits for EPTB Diagnosis.

  cutoff valuea (Abs@450 nm) AUC group (n = 197) positive results/total no. sensitivity, % specificity, %
TB CFP-1l0 kit 0.248 0.76 TB lymphadenitis (n = 36) 19/36 52.8  
non-TBLN (n = 161) 5/161   96.9
lymphadenitis (n = 25)
lymphoma (n = 14)
non-TB (n = 122)b
TB Ag85 kit 0.212 0.49 TB lymphadenitis (n = 36) 11/36 30.6  
non-TBLN (n = 161) 30/161   81.4
lymphadenitis (n = 25)
Lymphoma (n = 14)
non-TB (n = 122)b
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a

Cutoff values of both kits were established on the maximum Youden index value in a tested patient’s groups.

b

The non-TB control data are identical to the result shown in Figure 3.

So far, the MagPlas ELISA urine test appears to be a promising avenue for fast on-site TB diagnosis with high sensitivity, which provides a great potential for their use in longitudinal follow-up testing to monitor therapeutic efficacy. We hypothesized that an excreted antigen concentration intimately correlates with the virulence of Mtb, and thus antigen level profiling could reflect drug response in real time. Serial urine samples were collected from each TBLN patient (n = 9) during two or three distinct treatment visits and profiled with MagPlas ELISA kits. Figure 4e represents the changes in CFP-10 and Ag85 levels in urine after treatment. The initial antigen levels are varied across patients. We observed that five of nine cases (55.6%) showed a consistent decrease of urinary CFP-10 and ultimately reached an undetectable level post-therapy (patient nos. 2, 3, 7, 8, and 9). No significant CFP-10 level changes were observed in the other three of nine cases (patient nos. 1, 4, 6), and one case (patient no. 5) exhibited an elevated CFP-10 level after treatment. The reason for low correlation in these cases is unclear. However, we found that all of these subjects had a medical history of PTB infection in the past and consistently exhibited positivity in the IFN-γ release assays test (IGRA, QuantiFERON-TB Gold In-Tube test), which confirms their latent TB infection. Interestingly, one subject (patient no. 1) reported a recurrence of TB after treatment completion. But, at present, we could not conclude that there is a relationship between continuous antigen release and IGRA positivity because limited cases are tested in this study. Thus, further follow-up studies are necessary to understand the pathological signs and antigen secretion. In contrast, urinary Ag85 excretion was at a nondetectable level in most patients; only two of nine cases (22.2%) showed a significant decrease after treatment (patient nos. 1, 3).

In a series of clinical studies, we observed that the urinary CFP-10 level is highly correlated with diseased status and therapeutic efficacy. Unlike this, the urinary Ag85 levels are consistently lower than CFP-10 levels; particularly, it shows ineffective statistical values in TBLN diagnosis. The plausible reason is that the circulating Ag85 proteins prefer to form complexes with immunoglobulin and plasma fibronectin rather than in an unbound form.41 Thus, the Ag85 complex possibly shows a lower renal clearance albeit their sizes are small enough (30 to 32 kDa) to be readily cleared by the kidney, resulting in greater difficulty to be found in TBLN patient samples. Overall, the longitudinal follow-up data indicated that urinary CFP-10 antigen profiling could be used as predictor variables for TBLN treatment trajectories; however, we note that further study is required with larger and more frequent sampling to obtain statistical significance.

Conclusions

We proposed the MagPlas ELISA that enables fast (<3 h) and highly sensitive (<10 fM) biomarker detection. A clinical study with a total of 297 urine samples (i.e., 100 PTB, 36 EPTB, 39 lymphadenitis/lymphoma, and 122 non-TB control) revealed that urinary CFP-10 is a potent marker for noninvasive TB urine testing. The TB CFP-10 kit achieved robust sensitivity for PTB (85.0%) and EPTB (52.8%) patients with high specificity (96.7% and 96.9%). Meanwhile, the TB Ag85 kit showed lower sensitivity for PTB (47.0%) and was ineffective for EPTB cases, plausibly due to their lower renal excretion, thus only supportive for PTB urine testing. The longitudinal follow-up data of nine EPTB patients for 26 weeks indicated that urinary CFP-10 profiling could be used as predictor variables for treatment trajectories.

The MagPlas ELISA has many leading-edge features: (i) The MagPlas NZs integrate magnetic enrichment and catalytic amplification into a single reagent process. The colored signal can be straightforwardly read out with the naked eyes (up to pM range) and, if needed, quantified by a conventional plate reader (<10 fM of LOD). Note that this femtomolar detection sensitivity enables the monitoring of urinary CFP-10/Ag85 that is undetectable in conventional immunoassays. Our method represents >10-fold higher sensitivity than the highest level reported to date27,32 and also 3 orders of magnitude greater sensitivity than a commercial urine TB-IVD test (Alere Determine TB LAM Ag assay, Abbott Diagnostics;42Tables S5). (ii) Direct on-site TB diagnosis without culture or nucleic acid amplification (i.e., PCR) is a significant advance from current standard techniques. Culture or PCR processes usually have a potential risk of contamination and laboratory-acquired infection,8 therefore requiring sophisticated instruments and experts. In our approach, magnetic enriching minimized interference from crude urine molecules (e.g., creatinine, urea, uric acid) then transferred to enzymatic enhancement, consequently circumventing lengthy and cumbersome purification and amplification processes. (iii) Ultimately, the MagPlas ELISA is designed for direct use at the POC settings (Figure S16). The test outcome can be provided in less than 3 h, including sampling (0.5 h), bead-plate assay (2 h), and measurement (0.5 h), which is much shorter than the BD BACTEC MGIT culture system (∼2–6 weeks) and comparable to the Cepheid GeneXpert MTB/rifampicin (RIF) test (∼2–3 h). The assay cost could be estimated at under $3 per test (Tables S6), and color reader costs are ∼$300 or equipment-free, thus they are affordable worldwide to resource-limited areas.

The MagPlas ELISA showed high sensitivity comparable to the conventional PCR technique while retaining the simplicity and low cost of the ELISA concept, thus it could be a promising POC test for the diagnosis and management of active TB and possibly other emerging infectious diseases, such as severe acute respiratory syndrome (SARS), swine flu, Ebola, Middle East respiratory syndrome (MERS), and COVID-19.

Experimental Section

Synthesis of MagPlas Nanozymes

The Fe3O4 nanoclusters were synthesized using a one-pot solvothermal method according to a previously reported procedure.34 To synthesize Fe3O4–Au superparticles, i.e., MagPlas NZs, 20 mL of the Fe3O4 nanocluster solution (1 mg/mL) was treated with ultrasound (Sonics and Materials Inc. VC750, Newtown, CT) using a 5 s on/3 s off routine for 1 h. Then, Au coating was applied to the Fe3O4 surface following the method developed by Kim et al.21,22 The resulting solution was washed with deionized (DI) water to remove remnant precursors and resuspended in an aqueous solution (∼3.2 × 1010 particles/mL or ∼5.2 × 10–11 M), then stored at 4 °C until further use.

Magnetoplasmonic ELISA for Urine Test

Preparation of MagPlas Nanozyme Probes

The MagPlas NZ–antibody conjugates were prepared using a facile GBP-Au immobilization technique.22,27 In a typical assay, 3.6 mL of MagPlas NZs (5.2 × 10–11 M) solution was mixed with 1.5 mL of 0.1% BSA–PBS buffer (0.1% BSA in 0.1 M PBS pH 7.4), then 1 mL of 80 μg/mL GBP-antiCFP10 [G2] or GBP-antiAg85 [50B14] was added. Then, 4 mL of 0.1 M PBS (pH 7.4) was added and incubated at 27 °C for 30 min with shaking. Unbound antibodies were washed out three times by mixing with washing buffer (PBST: 0.1 M PBS pH 7.4 containing 0.05% (v/v) Tween 20). Washed nanoprobes were resuspended in 5 mL of blocking buffer (BSA-PBS), after 1 h shacking, washed three times with washing buffer. The final products were redispersed in 3.6 mL of PBS buffer for further use.

Preparation of MagPlas ELISA Plate

The silica-binding polypeptide (SBP) was used for direct immobilization of captures antibodies onto the 96-well microtiter plates. In brief, the PDMS prepolymer was mixed with a curing agent at a weight ratio of 10:1 and degassed in a vacuum desiccator for 1 h to remove the air bubbles. Then, 50 μL of the PDMS mixture was transferred to each well on the 96-well plate, thermally cured at 70 °C for 1 h, where a ∼1-mm-thick PDMS layer was fabricated (PDMS–96-well plate). Cured PDMS–96-well plate was washed with DI water and PBS (pH 7.4), and dried in a nitrogen stream. The plates were then coated with 100 μL of 120 ng/mL SBP-antiCFP10 [G3] or SBP-antiAg85 [8B3] in a shaking incubator at 27 °C for 30 min. After washing the plates three times with washing buffer (PBST), the antibody-immobilized surface was blocked with 100 μL of blocking buffer (BSA-PBS). After shaking at 27 °C for 30 min, the plates were washed three times with washing buffer.

MagPlas ELISA Urine Test

In a typical MagPlas ELISA urine test, 300 μL of MagPlas NZ probes (MagPlas NZ–GBP antibody conjugates, 5.2 × 10–11 M) was mixed with 300 μL of urine samples or 300-μL aliquots of serially diluted antigens at known concentrations, and followed by shaking at 37 °C for 1 h. After washing three times with washing buffer, target-captured MagPlas NZ probes were resuspended in 100 μL of PBS (pH 7.4), then transferred into antibody-immobilized 96-well plates (MagPlas ELISA plate), incubated by shaking at 37 °C for 1 h. The MagPlas probes bound plate was washed three times by PBS buffer, and subsequently, 200 μL of freshly prepared working solution (a mixture of TMB liquid substrate (Sigma-Aldrich T4444) and 0.5 M H2O2 in a volume ratio of 3:7) was added. After 10 min incubation at room temperature, 50 μL of stop solution (Sigma-Aldrich S5814) was added. The developed yellow color solution was aspirated after 5 min, stopped and transferred to a vacant 96-well plate, followed by reading the absorbance of each well at 450 nm using a microplate reader.

Clinical Samples

Urine samples were obtained from the Tuberculosis Specimen Biobank of Masan National Tuberculosis Hospital (Masan, Republic of Korea) and the Human Biobank of Chungnam National University Hospital (Daejeon, Republic of Korea). Of the 100 urine samples obtained from TB patients who were hospitalized for treatment, 90 patients were sputum smear and/or culture-positive and 10 patients were sputum smear and culture-negative for acid-fast bacillus (AFB) when the urines were collected. The control urine samples were obtained from 122 non-TB populations who were confirmed with chest X-ray and/or bacteriologic methods. Urine samples from extrapulmonary TB (TBLN, n = 36) and non-TBLN patients (lymphadenitis, n = 25, and lymphoma, n = 14) were collected at Dong-A University Hospital (Busan, Republic of Korea). TB diagnoses were confirmed by bacterial culture or biopsy with an RT-PCR test (Tables S3 and S4). Each sample was split into two aliquots and used for TB CFP-10 kit and TB Ag85 kit, respectively. The filtrates were used directly for antigen quantification with the MagPlas ELISA test. For longitudinal analysis, serial urine samples were collected from TBLN patients (n = 9) during two or three distinct treatment visits. The study protocol was approved by the Institutional Review Board (IRB) of the Chungnam National University Hospital (CNUH2015–08–003, CNUH2017–06–058) and the Dong-A University (IRB Protocol No. 14-157).

Characterization Tools

The UV–visible (UV–vis) spectra were recorded using a UV–vis spectrophotometer (model 8453, Agilent Technologies Inc., Santa Clara, CA), and particle size distributions and surface potentials were measured using a zeta-sizer (ZS Nano, Malvern Instruments Ltd., Worcestershire, UK). The morphologies and sizes of the MagPlas NPs were visualized using high resolution-transmittance electron microscopy (HR-TEM, JEOL, JEM-3010, Tokyo, Japan) and field-emission scanning electron microscopy (FE-SEM; S-4700, Hitachi, Tokyo, Japan). Furthermore, the X-ray diffraction (XRD) analysis was carried out using an X-ray powder diffractometer with Cu Kα radiation and a Ni filter (D8 FOCUS 2.2 kW, Bruker, Germany), and magnetic measurements were performed using vibrating sample magnetometry (VSM; model 6000, Quantum Design, Inc., San Diego, CA). The absorbance of samples in microtiter plates was read using a microplate reader (PerkinElmer Victor 3 1420 multilabel plate reader, Boston, MA).

Statistical Analysis

All measurements were performed in triplicate, and the data are presented as mean ± s.d unless noted otherwise. The measured antigen levels (CFP-10, and Ag85) were analyzed via logarithmic regression. All comparisons were assessed using a two-tailed Student’s t test, and P < 0.05 was considered statistically significant. In all box plots, measurement data (points) with median value (center line), 1%, 25%, 75%, and 99% value lines (box lines) are presented. The accuracy of the MagPlas ELISA tests was evaluated using the receiving operating characteristic (ROC) curves. The optimal cutoff points were determined using Youden’s index, which maximizes the sum of sensitivity and specificity. All diagnostic metrics (i.e., sensitivity and specificity) were calculated using standard formulas.

Acknowledgments

This study was supported by a grant from the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP; NRF-2019R1A2C2007825, NRF-2019K1A3A1A18116066, NRF-2019H1D3A1A01102565, NRF-2017R1A5A2015385).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00948.

  • Experimental details and supporting figures/tables for morphological and compositional characterization of MagPlas NZs; catalytic and magnetic property of MagPlas NZs; enzymatic efficiency of the MagPlas NZs; summary of antigens and antibodies; schematics of the procedure for preparing the MagPlas ELISA kit; clinical sample information; comparison of the sensitivity in urine-based Mtb detection assays; comparison of analytical performance with standard method; bill of materials for NZ synthesis and assay cost estimation (PDF)

The authors declare no competing financial interest.

Supplementary Material

oc1c00948_si_001.pdf (1.7MB, pdf)

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Supplementary Materials

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