Nanoparticle-Based Biosensing of Tuberculosis, an Affordable and Practical Alternative to Current Methods
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Instrumentation
2.3. Clinical Samples
2.4. Sputum Sample Processing
- Sputum Smear Microscopy (SSM): A smear was prepared by the Ziehl–Neelsen (ZN) method of the third (fresh) sputum sample following the standard protocol by the International Union Against Tuberculosis and Lung Disease [19] within 2 h after receipt at the hospital. Briefly, about 20 µL of fresh sputum sample was placed on a glass slide and heat fixed by a passing flame from a Bunsen burner. The slide was placed on a staining rack and 0.3% carbol fuchsin was poured over the smear. The underside of the slide was gently heated by passing a flame under the rack until fumes appeared. After cooling for about 5 min, the smear was rinsed with distilled water until no color appeared in the effluent. The smear was washed with 25% sulphuric acid several times until the smear appeared light pink in color. The smear was washed with distilled water and then the counter stain (0.3% methylene blue) was added to cover the smear. Distilled water was used to wash off the counter stain and then the smear was air-dried. Once ready, the smear was examined under a light microscope using 100× oil immersion objective to observe the presence of red-colored AFB.
- Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA): Decontamination reagent from the kit was added to the combined sputum sample in a 2:1 (reagent to sample) ratio in a 15-mL falcon tube, which was then manually agitated twice (or quick vortex) during a 15-min incubation period at room temperature. Subsequently, 2 mL of the decontaminated sputum sample were transferred to the test cartridge using a sterile disposable pipette (provided with the kit). The cartridge was loaded into the Xpert MTB/RIF PCR machine and operated for 1 hour and 50 min. At the end of the real-time PCR run, the result was generated [20].
- Nanoparticle-based Colorimetric Biosensing Assay (NCBA): About 1 mL of the decontaminated sputum sample was added into a 1.5 mL tube containing 100 µL of the nanoparticles. The GMNP and sputum were mixed and allowed to incubate for 5 min at room temperature. The tube was then placed in a magnetic rack to separate the magnetic GMNP-AFB complex and the supernatant was discarded. A smear was prepared by adding 20 µL of the concentrated GMNP-AFB complex on a glass slide following the SSM procedure, as described in Section 1 above.
2.5. Statistical Analysis
3. Results
4. Discussion
4.1. GMNP-AFB Complex
4.2. Novel Colorimetric Biosensing Mechanism
4.3. SSM vs. Xpert MTB/RIF
4.4. SSM vs. NCBA
4.5. NCBA vs. Xpert MTB/RIF
5. Conclusions
6. Ethics Approval
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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SSM Test | True TB Cases | Non-TB Cases | NCBA Test | True TB Cases | Non-TB Cases |
---|---|---|---|---|---|
Positive test | 32 | 0 | Positive test | 80 | 0 |
Negative test | 48 | 420 | Negative test | 0 | 420 |
Technique | Xpert MTB/RIF as the Gold Standard, % (95% CI) | ||||
---|---|---|---|---|---|
Sensitivity | Specificity | PPV | NPV | Accuracy | |
SSM Test | 40 (29–52) | 100 (99–100) | 100 | 90 (88–91) | 90 (87–93) |
NCBA Test | 100 (95–100) | 100 (99–100) | 100 | 100 | 100 (99–100) |
Xpert MTB/RIF Categories | Very Low | Low | Medium | High | Total |
---|---|---|---|---|---|
Xpert MTB/RIF | 10 | 22 | 29 | 19 | 80 |
NCBA | 10 | 22 | 29 | 19 | 80 |
SSM | 0 | 3 | 14 | 15 | 32 |
% Detection (NCBA/Xpert) | 100% | 100% | 100% | 100% | |
% Detection (SSM/Xpert) | 0% | 14% | 48% | 79% |
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Bhusal, N.; Shrestha, S.; Pote, N.; Alocilja, E.C. Nanoparticle-Based Biosensing of Tuberculosis, an Affordable and Practical Alternative to Current Methods. Biosensors 2019, 9, 1. https://doi.org/10.3390/bios9010001
Bhusal N, Shrestha S, Pote N, Alocilja EC. Nanoparticle-Based Biosensing of Tuberculosis, an Affordable and Practical Alternative to Current Methods. Biosensors. 2019; 9(1):1. https://doi.org/10.3390/bios9010001
Chicago/Turabian StyleBhusal, Nirajan, Sunaina Shrestha, Nisha Pote, and Evangelyn C. Alocilja. 2019. "Nanoparticle-Based Biosensing of Tuberculosis, an Affordable and Practical Alternative to Current Methods" Biosensors 9, no. 1: 1. https://doi.org/10.3390/bios9010001