Plastic-Chip-Based Magnetophoretic Immunoassay for Point-of-Care

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Plastic chip based magnetophoretic immunoassay for point-of-care diagnosis of tuberculosis Jeonghyo Kim, Minji Jang, Kyoung G. Lee, Kil-Soo Lee, Seok Jae Lee, Kyung Won Ro, In Sung Kang, Byung Do Jeong, Tae Jung Park, Hwa-Jung Kim, and Jaebeom Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06924 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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ACS Applied Materials & Interfaces

Plastic chip based magnetophoretic immunoassay for point-of-care diagnosis of tuberculosis Jeonghyo Kim1,†, Minji Jang1,†, Kyoung G. Lee2, Kil-Soo Lee3, Seok Jae Lee2, Kyung-Won Ro4, In Sung Kang4, Byung Do Jeong4, Tae Jung Park5, Hwa-Jung Kim6, Jaebeom Lee1,*

1

Departments of Cogno-Mechatronics Engineering, Pusan National University, Busan 46241,

Republic of Korea 2

Department of Nano Bio Research, National NanoFab Center (NNFC), Daejeon 305-806,

Republic of Korea 3

Department of Bacterial Respiratory Infections, Center for Infectious Diseases, National

Institute of Health, Korea Center for Disease Control and Prevention, Cheongju 28159, Republic of Korea 4

Scinco R&D Center, 746, Daedeok-daero, Yuseong-gu, Daejeon 34055, Republic of Korea

5

Department of Chemistry, Chung-Ang University, Seoul 06974, Republic of Korea

6

Department of Microbiology and Research Institute for Medical Science, College of

Medicine, Chungnam National University, Daejeon 35015, Republic of Korea †Both

contributed equally.

*Corresponding authors: Jaebeom Lee, PhD; E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract Tuberculosis (TB) remains a relevant infectious disease in the 21st century and its extermination is still far from being attained. Due to extreme infectivity of incipient TB patients, a rapid sensing system for proficient point-of-care (POC) diagnostics is required. In our study, a plastic chip−based magnetophoretic immunoassay (pcMPI) is introduced using magnetic and gold nanoparticles (NPs) modified with Mycobacterium tuberculosis (MTB) antibodies. This pcMPI offers an ultrasensitive limit of detection (LOD) of 1.8 pg•ml-1 for the detection of CFP-10, an MTB-secreted antigen, as a potential TB biomarker with high specificity. In addition, by combining the plastic chip with an automated spectrophotometer setup, advantages include ease of operation, rapid time to results (1 h), and cost-effectiveness. Furthermore, the pcMPI results using clinical sputum culture filtrate samples are competitively compared with and integrated clinical data collected from conventional tools such as the acid-fast bacilli (AFB) test, mycobacteria growth indicator tube (MGIT), polymerase chain reaction (PCR), and physiological results. CFP-10 concentrations were consistently higher in patients diagnosed with MTB infection than those seen in patients infected with non-tuberculosis mycobacteria (NTM) (P < 0.05), and this novel test can distinguish MTB and NTM while MGIT cannot. All these results indicate that this pcMPI has the potential to become a new commercial TB diagnostic POC platform in view of its sensitivity, portability, and affordability.

Keywords: Tuberculosis (TB), magnetophoretic immunoassay (MPI), Mycobacterium tuberculosis (MTB), CFP-10, plastic chip, mycobacteria growth indicator tube (MGIT)


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1. Introduction Tuberculosis (TB), a neglected infectious disease, took possession of an estimated 9.6 million new cases and resulted in 1.5 million deaths in 2014.1 TB can quickly spread internationally and is difficult to prevent because it spreads through the air. In particular, early diagnosis techniques are critical to strengthen care and diagnosis of incipient TB patients. Traditional diagnosis processes such as bacterial culture, microbiological, molecular biological, and immunological diagnosis methods have presented crucial barriers for preventing proliferation of the disease because these methods are still exorbitant, laborious, lengthy, and instrument-dependent. Current techniques are not easy to implement for routine clinical use, especially in developing countries, which make up most of the red alert areas for epidemic diseases.2 The emerging field of nano/microtechnology, which can surpass the limits of traditional sensors in sensitivity, portability, and affordability, has developed manifold diagnosis platforms and methods. But technological reviews inform us that current TB diagnosis methods are limited by three primary limitations: low specificity, long reaction time for diagnosis, and most of all, high cost for reagents and analysis.3 For example, the Mycobacterium tuberculosis (MTB) culture test, which remains the standard laboratory diagnosis method for active TB disease and identification of drug-resistant strains, requires 10−40 days of processing time including 28 days for MTB cultivation, which does not meet the demand for rapid detection.4-6 Recently developed polymerase chain reaction (PCR) based technologies reduce the delays in obtaining results to days rather than weeks, however still it have constraints toward point-of-care (POC) diagnostics, such as high costs and need for sophisticated instrumentation.3 The clinically used mycobacteria growth indicator tube (MGIT) is designed for rapid



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detection of mycobacteria in liquid culture of clinical specimens, and is based on oxygen consumption of actively growing mycobacteria in the sample, which is reflected by changes in fluorescence intensity.7-8 It is currently the gold standard diagnostic, is the easiest and most reliable sensing tool for monitoring MTB, and most hospitals adopt MGIT to monitor infections in TB patients. However, an inherent drawback of this method is its inability to distinguish whether the increase in oxygen consumption is from active MTB or nontuberculosis mycobacteria (NTM) in the sputa of patients. PCR based methods has been used as a complementary strategy, but PCR analysis cannot differentiate between latent and active TB specimens.9-10 However, immunoassays have a unique strength in active MTB monitoring since only live MTB bacteria secrete antigens. In particular, in early stages of TB, CFP-10, Ag85, and HspX represent the majority of antigens.11-12 Moreover, immunological TB diagnostic assays are prominent approach for the development of POC testing tools because which allow cost-effectiveness, easy to miniaturization, and high portability, and disposability. Since the majority of the cost of a biosensing kit is from bio-moieties (e.g. antibodies), a reservoir with a minimal level of sample volume is important for developing a cost-effective sensing kit. Furthermore, novel TB sensors are likely to utilize biological fluids such as blood, urine, and sputum directly with minimal pretreatment, since pretreatments become laborious and decrease accessibility. For example, recently our laboratory reported a novel sensing system using magnetic and gold nanoparticles (NPs) to monitor TB antigens,13-14 where TB was monitored by a sandwich assay-like mechanism using two different probe antibodies. After coupling between probes and analytes, color change could be easily monitored. However, its major drawback was a minimum sensing volume of 1.5-3 mL due to the geo-optical limitation of the used cuvette and absorbance spectrophotometer, where the volume of sample holder directly corresponds with consumption of immunomoieties and nanomaterials. Notably, low volume


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negatively affects the limitation of detection (LOD) because colorimetry corresponds with the beam path (l) of a sample solution according to the Beer-Lambert law (A=ɛcl). Colorimetry is believed to be an affordable sensing method since it can be monitored by a simple spectrophotometer or even without any instrumentation. Therefore, in order to utilize a minimal amount of sensing components, and a combination of two sensing systems (immunoassay and colorimetry), a novel plastic chipbased magnetophoretic immunoassay (pcMPI) analysis was designed for detecting the CFP10 antigen. The volume of the chip reservoir was decreased as much as possible (down to 400 µL) for monitoring with colorimetry, and the beam path of the incident light in a newlydesigned spectrophotometer was similar to the conventional cuvette. When using the novel pcMPI and spectrophotometer, the consumption of bio-moieties decreases more than 200% by using a smaller sensing volume, while maintaining sensitivity (1.8 pg•mL-1) comparable to conventional systems. Furthermore, in the sensing test with clinical specimens of MGIT, pcMPI was highly competitive. Together, the results show that this unique pcMPI has the potential to become a new platform for point-of-care (POC) TB diagnostics.



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2. Experimental details 2.1 Materials and instruments Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O, 99.9%), trisodium citrate dihydrate (Na3C6H5O7·2H2O, >99.0%), gallic acid, and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (St. Louis, MO). Isoflavone was extracted from commercial soybeans. Carboxyl-functionalized magnetic microparticles (MMPs, 1 µm diameter) were purchased from Invitrogen (Carlsbad, CA) and used according to the manufacturer’s protocol. Phosphate buffered saline (PBS, 1 M, pH 7.4) was obtained from BD Biosciences (San Jose, CA) and diluted to 0.01 M, pH 7.4 prior to use. The morphologies and sizes of magnetic/plasmonic probes were characterized by transmission electron microscopy (TEM) (JEM-2100F, JEOL, Ltd., Tokyo, Japan), ﬁeldemission scanning electron microscopy (FE-SEM) (S-4700, Hitachi, Tokyo, Japan). Their surface potentials and particle size distributions were monitored using a Zetasizer (ZS Nano, Malvern Instruments, Malvern, U.K.).

2.2 Preparation of CFP-10 antigens and antibodies Recombinant CFP-10 protein and HspX were expressed in Escherichia coli (E. coli) and purified according to previously reported methods.15 The native antigen 85 complex, called Ag85, was purified from MTB culture filtrate by using a procedure described by Lee et al.16 The gold-binding protein (GBP)-fusion antibodies against CFP-10 antigens were prepared following the previously reported method of Kim et al.17-18 Briefly, DNA genes encoding the 6 histidine-tagged gold-binding protein (6His-GBP) and the CFP-10 [G2] and [G3] were amplified by polymerase chain reaction (PCR). The products of PCR were digested with NdeI


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and NcoI, and then ligated into same sites of the pET-22b(+) expression vector. The E. coli BL21(DE3) was used as a host strain for the expression of 6His-GBP-CFP-10 [G2] and [G3]. Recombinant E. coli BL21(DE3) strains were cultivated in Luria-Bertain (LB) medium with 100 µg/ml of ampicillin at 37°C and 200 rpm, and after lysis procedure by ultrasonication, and then proteins were purified by AKTA purifier (GE Healthcare Bio-Science, Piscataway, NJ) using Ni column. Two different types of CFP-10 antibodies, [G2] and [G3], were used for the sandwich-type immunosensing. 2.3 Preparation of magnetic/plasmonic probes 2.3.1. Au NP probes The procedures for Au NP synthesis and their conjugation with antibody (GBP-antiCFP-10 [G2]) were as follows: Au NPs were prepared according to a previously reported protocol.19 The synthesized Au NPs had an average diameter of 50 nm with less than 10% variation in size distribution. To prepare the Au NP probes, 8 mL of Au NP solution (1.10 × 10-9 M) was mixed with 100 µL of GBP-antiCFP-10 [G2] solution (32 µg•mL-1), and incubated for 40 min at room temperature with gentle shaking (150 rpm). Then 700 µL of BSA (0.1% w/v in DI water) was added for 1 h at room temperature to block nonspecific binding sites. The asprepared G2-Au NP Probes were filtered with a 0.22-µm hydrophilic syringe filter and stored in a 4°C refrigerator until use. 2.3.2. MMP@Au probes Au island-deposited MMP (MMP@Au) probes were prepared by following our previously reported protocol with slight modifications.20 Then 100 µL of GBP-antiCFP-10 [G3] solution (255 µg•mL-1) was added to the MMP@Au NP solution. The mixed solution was resuspended in 2 mL of PBS and incubated for 40 min at room temperature with gentle



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shaking (150 rpm). Unbound G3 antibodies were washed away twice in PBS. After the conjugation process, NP regions not bound to antibody were blocked with 1 mL of PBS containing 1% w/v BSA, then were washed three times. The G3-MMP@Au probes were resuspended in 8 mL of PBS and stored in a 4°C refrigerator until further use. 2.4 Design of plastic chip The flat cyclic olefin copolymer (COC) plate was fabricated by a microinjection molding machine (A270C 400-100, ARBURG) with a flat nickel (Ni) stamp as previously described. The device is composed of two layers of COC chips, three layers of adhesive film, and two layers of plastic film. The adhesive film is mainly composed of well-known polyimide (PI) with coating of silicone-based adhesive layers on both sides. The detailed dimensions of the device and fabrication processes are described in Figure 2 (A). Overall chip dimensions are 20 × 30 × 4 mm3. Two COC-based chips were directly prepared using a micro milling machine. This machine is equipped with a 24,000 rpm spindle motor which rotates the end mills. Both square end mills (0.5, 1, and 2 mm normal diameter) and the ball nose end mill (1 mm normal diameter) were used to create the desired chambers and structures. The spindleattached motor heads were carefully controlled by a computer using G-code programming (Visual Mill). After COC chips were created, two layers of film were attached to the top and bottom of the chip using adhesive films with same dimensions as the chips. The top layers of film have inlet and outlet holes for liquid injection.

2.5 Procedure of plastic chip based magnetophoretic immmunoassay (pcMPI) with a unique design of spectrophotometer A typical pcMPI procedure is composed of two steps. Step 1: the assay is initiated by


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mixing 200 µL of MMP@Au probe solution (375 µg•mL-1) and 200 µL of Au NP probes with 10 µL of target solution (CFP-10 antigen or pretreated clinical samples) in a designated plastic chip. The mixture in the plastic chip is allowed to incubate under gentle shaking (150 rpm) at 37°C for 30 min. The Au NP probes were specifically bound to MMP@Au probes via an antigen-antibody reaction. Step 2: Sandwich-structured nanocomposites, i.e., [AuNPsG2 probe]-CFP10 antigen-[G3-MMP@AuNPs probe] were then magnetically separated from the solution and absorbance of the supernatant was observed using a UV-Vis spectrophotometer (Scinco Nano-MD, Scinco Co., Seoul, South Korea) which was specially designed for MPI, including a sample mixer and collector via an external magnet (See Figure 2 (B)). 2.6 Preparation of clinical MGIT samples for comparison with pcMPI A clinically used MGIT system was utilized for comparison with our pcMPI system. A total of 16 acid-fast bacilli (AFB) smear positive sputum samples were cultured in MGIT vials according to the manufacturer’s protocol and culture supernatants were obtained from MGIT test vials on the day of the positive signal (See Table S1). Then the obtained culture filtrates were diluted 1:2 with pH 7.4 PBS and filtered once again with a 0.22-µm hydrophilic acetate filter (SLGP033NS, Millipore Co., Bedford, MA) to exclude any unwanted remnants in culture media.

3. Results and Discussion 3.1 pcMPI diagnostics kit Figure 1 illustrates the entire procedure from clinical sample preparation to pcMPI



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based TB diagnostics. To validate the potential utility of the pcMPI kit as a clinical diagnostic tool, we analyzed clinical sputum culture filtrates (CFs) as a target clinical matrix. Sputum samples were collected from a total of 16 suspected TB cases, then underwent a standard bacteriological evaluation using a liquid culture system (MGIT). When a positive signal was flagged from MGIT, culture supernatants were aspirated from the MGIT test tube and evaluated with the pcMPI (Figure 1 (A)). The proposed pcMPI procedures are shown schematically in Figure 1 (B), which were composed of four simple steps: sample injection, mixing and target capture, magnetophoresis, and plasmonic signal quantification, which were all carried out in a plastic chip module. In steps 1 and 2, a target sample with both Au NP and MMP@Au probes were injected into the plastic chip simultaneously. Then, the loaded components were mixed and target antigens were captured by immunoreaction. Since GBPantiCFP-10 [G2] and [G3], which were attached on the surface of 2 different NP probes, can recognize different regions of the CFP-10 antigen, these probes can link with CFP-10 antigens to form sandwich-structured immunocomplexes. These sandwich structures can be removed from the detection window by external magnetic force. In our experiment, a handheld neodymium-iron-boron (NdFeB) magnet was used (5.52 × 104 Gauss.) for magnetophoresis. Then, the absorbance of the remnant Au NP probes was inversely correlated to the quantity of target antigen in the specimen. The quantitative analysis of plasmonic signal was carried out in this manner.


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Figure. 1. Schematic illustration of plastic chip - based magnetophoretic immunoassay (pcMPI). (A) Procedure for the preparation of clinical samples. (B) Procedure for quantification of CFP-10 TB biomarkers using pcMPI kit-based diagnostics.

In our previous work, the magnetophoresis process was performed in a micro cuvette (104.002-OS, path length 1.0 cm, Hellma GmbH, Müllheim, Germany) with a reaction reservoir of 800 µL, and an MPI analysis separation time of 10 min is required to reach magnetophoretic saturation (Figure S1).13 In the current study, two major achievements have been accomplished with this sensing system: effective design of the magnetophoresis reservoir and use of a colorimetric spectrophotometer. In our newly designed plastic chip, the detection window is enlarged to 81 mm2 (Figure 2 (A)) while the detection window of the previous micro cuvette was only 10 mm2; the incident beam can pass through an 8-fold larger area in our new chip. Furthermore, the immune-reacted complexes of antigens and NP probes were magnetically collected in the upper area of the cuvette in the previous system, where the maximum magnetophoretic distance for separation and optical sensing was 25-35 mm (same as the z-height of the micro cuvette) and this took at least 10 min to complete. In the pcMPI



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system, the maximum distance for immunocomplex collection was decreased to 9 mm since the complexes are collected on the side wall. Due to these geometrical considerations of reservoir design, the magnetophoretic separation process can be completed within 1 min, or even faster when two or four magnets are loaded in each wall (unpublished results). Meanwhile, a new spectrophotometer was designed for magnetophoresis, and the automated magnetic holder accessory is adapted for convenient use and optimum performance of MPI assays (Figure 2 (B)). The specialized holder accessory is composed of three distinct functional regions for sample mixing, magnetic separation, and signal measurement. The chip holder is able to move laterally through the entire range of the accessory by precision stepper motors. The procedure of magnetophoresis with the designated accessory is as follows (Figure 2 (B-b, c, d)): ① The sample-loaded chip is mounted on a holder and moved to the mixing zone for sample mixing (horizontal vibration, 60 rpm, 30 min). ② Magnetophoresis is initiated by moving the chip to the magnetic separation zone, ③ where the chip is placed directly onto the NdFeB magnet plate (5.52 × 104 Gauss, 1 min). ④ After magnetophoresis, the motorized holder moves the chip to the detection line, and ⑤ the UV-Vis beam measures the absorbance of the sample. This newly developed spectrophotometer setup suggests a prototype for automated MPI analysis with a plastic chip. Since the operation is managed by fully programmed software and hardware, users just inject sample into the plastic chip containing both probes, then one-click diagnostics can be performed and results are provided within 1 h.


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Figure 2. The plastic chip and automated spectrophotometry for pcMPI. (A) Detailed dimensions and fabrication process of the plastic chip prototype used for MPI analysis. (B) Photography of the specialized spectrophotometer design used for magnetophoresis. Layout of automated magnetic chip holder (a) and the procedures of pcMPI diagnostics with the accessory (b, c, d).

3.2 Characterization of magnetic/plasmonic probes. Au NPs were synthesized in an aqueous solution through the reduction of HAuCl4 using trisodium citrate, and then were functionalized with GBP antibody [G2].19 The as-prepared Au NP probes had an average diameter of 54.7 ± 5.3 nm with 9.6% relative standard deviation (RSD%) based on 250 counts in a TEM image (Figure 3 (A)). The bioconjugation of Au NPs was further confirmed by analysis of UV-vis spectra taken during probe preparation. As shown in Figure 3 (C), 55 nm Au NPs have a surface plasmon resonance (SPR) band at a wavelength of 535 nm that sequentially shifted to 537 nm and 540 nm following G2-antibody immobilization and BSA-surface blocking. The shift of the center peak position to a longer wavelength with a limited change in Gaussian band shape indicates



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that the proteins are stably immobilized on the Au NP surface without any aggregation.21 Since MPI monitors differences in target antigen concentration based on the integrated value of the plasmonic absorbance band, it is crucial not only to verify the stability of Au NPs during analysis but also to avoid any uncontrollable peak shifts, which are usually observed due to protein aggregation or change in nanostructure surface charge. In our experiments, all probes have been shown to remain stable during the MPI process by carefully monitoring zeta-potential variance and peak wavelength shifts.22 Also the surface chemistry for biomoiety conjugation has to be tightly controlled, so in our experiments a designated volume of BSA (and other short chemicals, e.g., polyethylene glycol if necessary) was attached to avoid any irrelevant adsorption of organic compounds during the MPI analysis. In order to take advantage of a GBP antibody, we designed a noble probe nanostructure. Au NPs were intentionally spread uniformly on the surface of large MMPs instead of making a shell structure, since Au NP shells can attenuate walls of magnetic flux of internal magnetic materials. For example, Fe3O4@Au NPs that were previously developed in our laboratory take 8-10 h to separate in a 4 mL plastic cuvette using a NbFeB magnet, which is much slower than 4-5 h for Fe3O4 without Au shells.23-24 With partial Au shell coverage, magnetic properties of MMPs were preserved (this will be further discussed in Figure 4). Then, GBP antibodies [G3] were immobilized to the surface of the MMP@Au NPs directly by means of a GBP-Au specific interaction.17 Figure 3 (B) presents representative TEM images of the prepared MMP@Au probes and size distribution curves of decorated Au islands on the surface of MMPs. Commercial MMPs approximately 1.05 µm in diameter (Dynabeads® MyOneTM Carboxylic Acid, Invitrogen, Carlsbad, CA) were used as core magnetic particles (Figure S2). The Au islands were deposited with an average diameter of 54.9 ± 8.7 nm (based on 100 counts, inset in Figure 3 (B)). Notice that the size of the MMP@Au probes reaches a semi-micrometer scale, which is intentional to speed up the separation process. The


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MMP@Au probe solution has a burgundy color, and is darker than Au NP solution due to strong absorbance of the relatively large MMPs. Indeed, the probes readily separated by applying external magnetic force, and the color of the solution was transparent within 1 min (inset in Figure 3 (D)), which reflects the rapid separation of antigen-binding probes in the MPI analysis. In UV-vis spectra, broad absorbance intensity from the MMP@Au probes completely vanished to an optical density (O.D.) lower than 10-5 in the range of 400 to 800 nm (Figure 3 (D)).

Figure. 3. Characterization of magnetic and plasmonic probes. Representative TEM images of (A) Au NP probes and (B) MMP@Au probes. Insets in (A) and (B) show the size distribution of synthesized bare Au NPs and deposited Au islands on the surface of MMPs, respectively. (C) Comparison of UV-Vis spectra for (1) bare Au NPs (black solid line), (2) G2 antibody-conjugated Au NPs (G2 Ab-Au NPs, red dashed line), and (3) BSA-blocked G2-Au NPs (BSA-G2 Ab-Au NPs, blue



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dots). (D) UV-Vis spectra and photographs (inset) to demonstrate magnetic separation using MMP@Au probes in 1 min.

The magnetic separation of MMP@Au probes in the solution should be as rapid as possible for magnetophoretic assay. To investigate the influence of the Au shielding effect on the magnetophoretic mobility and rate of MMP@Au NPs, the time-dependent absorbance profiles and colloidal stability were characterized. The decrease of magnetization seen in Fe3O4 cores after coating with Au shells has already been discussed in many reports, and has been mainly understood by inter- and intra-particle interactions.25-26 Firstly, addition of Au shells onto a Fe3O4 surface increases the inter-particle spacing between magnetic cores, resulting in a decrease in the coupling of magnetic moment. Simultaneously, the intra-particle system magnetization response is also influenced by interfacial stress and competing anisotropies at the direct contact of two different magnetic phases, thereby attenuating the wall of magnetic flux. Attaching the Au shells on the magnetic cores is the best choice for easy and highly efficient antibody conjugation, however it has possibility to decrease separation speed. To avoid loss of magnetic properties, firstly, Au shells of nanometer scale were deposited on the cores of micrometer scale (core to shell thickness ratio = 20:1), and Au NPs did not perfectly cover the core, which can minimize the loss of inter-particle coupling. Secondly, the bare MMPs are coated with cross-linked polystyrene and glycidyl ether, creating a gap between the core and the shells, thereby reducing the direct interaction between the magnetic moment and the plasmonic counterpart in the intra-particle system. The magnetophoretic mobility and rate for MMPs are shown in Figure 4 before and after partial coating with Au shells. NdFeB magnets were positioned underneath the plastic chip containing equal concentrations of MMPs or MMP@Au NPs (40 mg/mL) and the absorbance at 550 nm was measured every 5 sec during magnetophoresis. Figure 4 (A) shows that


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MMP@Au NPs separated slightly faster than MMPs at the beginning of separation, but both curves achieved the same asymptotic line within 2 min (real-time monitoring of the magnetophoresis process is provided in Figure 4 (B) and Movie S1). The rate of magnetophoresis, defined as absorbance change per sec (abs/s), was also compared (Figure 4 (A) inset). From 0-10 s, the magnetophoretic rate of MMP@Au NPs was 1.5 times faster than bare MMPs, but rates of both samples steadily decreased after that initial time period. To clarify the stability of dispersions, dynamic light scattering (DLS) analysis was employed (Figure 4 (C)). The zeta potential of MMPs was -53.6 mV and it increased to -44.6 mV after being covered with Au shells. Since colloidal dispersion surface potentials greater than ±40 mV are believed to be electrostatically stable, and size distribution also does not show an aggregation peak (Figure S3), the colloidal stability of MMP@Au NPs was deemed acceptable. Therefore, it is probable that the faster magnetic separation of MMP@Au NPs does not result from NP aggregation, but from the inhibition of magnetophoresis in MMPs colloidal solution due to the particle-to-particle repulsion because of the relatively higher surface charge of bare MMPs. From these results, magnetic properties were considered to be preserved in MMP@Au NPs without any loss of magnetophoresis velocity or colloidal stability.



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Figure. 4. Comparison of magnetophoretic mobility and rate for MMPs before and after partial coating with Au shells. (A) Time-based UV-vis absorbance kinetic plot for MMPs (black squares) and MMP@Au NPs (red circles). Inset shows the 1st order differentiation plot based on normalized absorbance. (B) Photographic images during magnetophoresis. NdFeB magnet is located underneath the chip and the movement of MMPs and MMP@Au NPs are indicated by arrows. (C) Comparison of zeta potential for MMPs and MMP@Au NPs.

3.3 Sensitivity and selectivity of pcMPI system. In the pcMPI analysis, the final absorbance intensity of Au NP probes after magnetophoresis was inversely proportional to the concentration of target antigen, which means that the amount of target detection can directly be expressed as delta absorbance (∆ABS), the difference in absorbance between the control (ABS0) and target injected sample (ABSS). To comparatively express absorbance change, ∆ABS values were normalized to


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ABS0, which is given by n∆ABS as determined by the following equation (1).

% = − × 100 % =

× 100 %

(1),

where ABS0 stands for the absorbance of control sample (without CFP-10), ABSS stands for the absorbance of each sample (with CFP-10), and all absorbance values were measured at 540 nm (peak wavelength of Au NP probes). For the quantification of CFP-10 antigen, a calibration curve was obtained with respect to the n∆ABS and it shows a logarithmic linear relationship with a series of standard CFP-10 concentrations ranging from 5.1 × 10-12

to 5.1

× 10-7 g•mL-1 (Figure 5 (A)). The regression equation for this curve is Y = 0.9013 ln(X) + 26.69 (R2 = 0.9751, n=3), where Y is n∆ABS and X is CFP-10 concentration. The limit of detection (LOD) in this sensing system was determined to be 1.8 × 10-12 g•ml-1 (1.8 × 10-13 M) and the threshold signal for the pcMPI was determined by the signal at limit of detection, SLOD, which is 2.3% (The details about LOD calculation are described in supporting methods). A selectivity test was performed using equal concentrations (0.5 µg•mL-1) of CFP-10 antigen and other nonspecific proteins, streptavidin (SA), bovine serum albumin (BSA), insulin, HspX, and Ag85 TB antigen. In particular, HspX and Ag85 were selected for the selectivity test in order to rule out possible cross-reactivity of G2 and G3 antibodies before clinical evaluation since they are known early stage secretory antigens in M. tuberculosis infections.27-28 Albumin is the most abundant protein in the blood, therefore it was chosen to validate further clinical accessibility of pcMPI. Figure 5 (B) shows the result of the selectivity tests, the signals from CFP-10 sample showed higher ∆ABS@540 nm compared with the nonspecific antigen and protein samples. There were some fluctuations of absorbance values within 20% that are likely related to inevitable light scattering originating



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from electrostatic, hydrogen, or van der Waals interactions inherent in a highly concentrated biological sample.

Figure. 5. Sensitivity and selectivity of the pcMPI system. (A) Calibration curve for the detection of CFP-10 at concentrations ranging from 5.1 × 10-12 to 5.1 × 10-7 g•mL-1. The n∆ABS value indicates the change in normalized absorbance between without target and with target, the threshold line is determined by the signal at limit of detection (SLOD). (B) Specificity of pcMPI for CFP-10. Various proteins (Ag85, HspX, bovine serum albumin, insulin, and streptavidin) at equal concentrations (0.5 µg•mL-1) were used as non-target control groups. All experiments were performed in triplicate.

3.4 Clinical utility of pcMPI system To evaluate the clinical utility of the pcMPI platform, a blind test was carried out using 16 clinical sputum culture filtrates (CFs); 11 MTB-positive samples from TB patients and 5 MTB-negative samples from non-tuberculosis mycobacteria (NTM)-infected patients that were used as negative controls. The patients’ MTB-positive or MTB-negative status was determined by medical doctors based on integrated data collected from conventional tools such as AFB test, MGIT, PCR, and physiological results (Table S1). The result of the blind test is shown in Figure 6. The CFP-10 concentration in the sputum CFs for each patient was estimated by comparing the n∆ABS value from the pcMPI analysis with the calibration curve


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in Figure 5 (A). We chose CFP-10 as a target antigen because CFP-10 is a protein that is abundantly secreted into the extracellular environment by MTB-specific immune responses,29 but is absent from most NTM-containing culture media.30 Therefore, CFP-10 antigen is a promising candidate biomarker for distinguishing MTB from NTM. Rapid discrimination between MTB and NTM infection is important for determining appropriate treatment and controlling TB prevalence, nevertheless current smear tests and liquid culture-based standard methodology (i.e. MGIT) have failed to identify NTM species.31 Assays performed on NTM infection samples showed consistently lower levels of CFP-10 than MTB infection samples. The average CFP-10 concentration of NTM and MTB groups are 5.6 fg/mL and 88.3 pg/mL, respectively (statistically different as determined from Student’s t test, P < 0.05). The highest CFP-10 concentration in the NTM infected group was ~10 pg/mL, and most of the CFP-10 concentrations in the MTB infected group were >10 pg/mL. Only three cases diagnosed with MTB infection overlapped with the NTM infected group. These three MTB infected cases could be false negatives in our assay; the main plausible reason for these outliers is the heterogeneity of antigen recognition in clinical specimens which could result from multiple factors. The quantity of secretory mycobacterial antigens present in the sputum specimens could vary greatly due to the immunogenetic background of the infected host,32 different stages of disease, and bacillary load in the sputum,33 resulting in variation of antigen secretion between individuals. In addition, in some cases, antigenic epitopes in sputum could be masked by body fluids, making the antigens unavailable for conjugation with antibody.34 These factors could interfere with the low assay sensitivity of the assay and could contribute to false negative results. While there are limitations attributed to the complexity of clinical assessment, this can likely be improved by merging the assay with multiplex antigen detection.35 Since clinical sputum specimens contain several M. tuberculosis-secretory antigens, i.e. Ag85, ESAT-6, and HspX, the simultaneous detection of multiple antigens



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could decrease the likelihood of false negative results. Currently, based on this idea, further development of pcMPI with multiplex antigen detection in clinical samples has been attempted to improve diagnostic reliability. This will be discussed in later publications (unpublished data). In recent studies, magnetophoretic assay (MPA) strategies have been applied to detect diverse target markers for quantitative analysis. For example, we previously applied MPI to identify the CFP-10 antigen in MTB culture and demonstrated a linear relationship between the concentration of secreted antigen in culture supernatants and MTB culture time.13 MPAs have also been applied to quantitation of DNA for the monitoring of genetic mutations in fishery products.24 More recently, a low-cost transducer was developed for magnetophoretic immunosensing applications utilizing laser diodes, a solar cell, and a multimeter.36 Additionally, Chen’s group demonstrated a new visual nucleic acid detection method based on Mie scattering of polystyrene microparticles (PMPs) and magnetophoretic effect,37 they also developed an enzyme-free amplification method using Au nano sticky balls which reached 10 attomoles of ssDNA detection.38 But to our knowledge, our work is the first attempt to apply an MPA strategy for detecting a target biomarker in clinical specimens with an automated POC diagnostics system. We believe that this system could be applied to many other in vitro diagnostics or to hazardous materials detection.


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Figure. 6. Clinical sample test with pcMPI system. Scatter plot of secretory CFP-10 antigen levels in clinical sputum culture filtrates from MTB-infected (positive) and NTM-infected (negative) patients. The SD from triplicates for each test case are shown and statistical significance was determined using a Student’s t test (*P < 0.05).

4. Conclusion In this study, we developed a fully integrated POC system for detection of the marker protein CFP-10 to diagnose TB infection based on a pcMPI strategy. Two different types of antibodies, GBP-antiCFP-10 [G2] and [G3], were modified with Au NPs and MMP@Au NPs respectively; these two nanoprobes can link together through the CFP-10 antigen. The sandwich-structured nanocomplex was rapidly separated from the assay solution by application of an external magnetic field, resulting in a change of solution turbidity that can be monitored. By combining a plastic chip and automated spectrophotometer setup, the pcMPI allows a simple and convenient immunoassay with an ultrasensitive LOD level of 1.8



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pg•ml-1 and high specificity. Clinical validation was performed on 16 clinical sputum CFs and the results show that the pcMPI can be used to measure CFP-10 concentration in sputum CFs at clinically relevant concentrations. Importantly, this approach of detecting a secretory antigen can differentiate between MTB and NTM infections to help clinicians make appropriate treatment decisions where standard liquid culture diagnostic tools fail. The pcMPI based POC diagnostics offer three principal advantages over conventional immunebased diagnosis techniques: (1) The pcMPI provides user-friendly measurement of the target biomarker with simple two-step operation and a completely automated workflow system. (2) Users can get final quantitative results in hours rather than days. By comparison, conventional ELISA needs at least a few hours of signal developing time, including cumbersome washing steps, magnetic separation, and enzyme reaction. But the proposed method affords a wash-free immunoassay with a 1-min signal transducing process, and the entire procedure can be completed within 60 min. (3) The pcMPI is composed of inexpensive materials: disposable plastic chips and reagents (two types of nanoprobes) cost

Plastic-Chip-Based Magnetophoretic Immunoassay for Point-of-Care

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