Fast and Highly Sensitive Detection of Pathogens Wreathed with

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Fast and Highly Sensitive Detection of Pathogens Wreathed with Magnetic Nanoparticles Using Dark-Field Microscope Fenglei Chen, Fang Tang, Chih-Tsung Yang, Xinyao Zhao, Jun Wang, Benjamin Thierry, Vipul Bansal, Jianjun Dai, and Xin Zhou ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00785 • Publication Date (Web): 23 Sep 2018 Downloaded from http://pubs.acs.org on September 24, 2018

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ACS Sensors

Fast and Highly Sensitive Detection of Pathogens Wreathed with Magnetic Nanoparticles Using Dark-Field Microscope Fenglei Chen†, Fang Tang‡, Chih-Tsung Yang§, Xinyao Zhao║, Jun Wang║, Benjamin Thierry§, Vipul Bansal¶, Jianjun Dai‡, and Xin Zhou*,† †

College of Veterinary Medicine, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China

‡

Key Laboratory of Animal Bacteriology, Ministry of Agriculture, College of veterinary medicine, Nanjing Agricultural University, Nanjing 210095, China

§

Future Industries Institute and ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Mawson Lakes Campus, Mawson Lakes, South Australia 5095, Australia ║

School of Biological Science and Medical Engineering, Southeast University, Nanjing 210009, China



Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Science, RMIT University, Melbourne, VIC 3001, Australia KEYWORDS: pathogens detection, dark-field microscope, magnetic nanoparticles, cryptosporidium parvum

ABSTRACT: Cryptosporidium parvum (C. parvum) is a highly potent zoonotic pathogen, which can do significant harm to both human being and livestock. However, existing technologies or methods are deficient for rapid on-site detection of water contaminated with C. parvum. Better detection approaches are needed to allow water management agencies to stop major breakout of the pathogen. Herein, we present a novel detection method for cryptosporidium in a tiny drop of sample using magnetic nanoparticle (MNP) probe combined with dark-field microscopy in 30 min. The designed MNP probes bind with high affinity to C. parvum, resulting in the formation of a golden garland-like structure under dark-field microscopy. This MNP-based dark-field counting strategy yields an amazing PCR-like sensitivity of 8 attomolar (aM) (5 pathogens in 1 μL). Importantly, the assay is very rapid (~30 minutes) and is very simple to perform as it involves only one step of mixing and magnetic separation, followed by dropping on a slide for counting under dark-field microscope. Combining the advantages of the specific light-scattering characteristic of MNP probe under dark-field and the selective magnetic separation ability of functionalized MNP; the proposed MNP-based dark-field enumeration method offers low cost and significant translational potential.

Cryptosporidium parvum (C. parvum), one of the major zoonotic parasitic protozoans, is a significant public health concern and a major cause of potentially severe human and livestock diarrhea called Cryptosporidiosis.1-3 C. parvum is transmitted to human mainly through gastrointestinal infections due to the consumption of food or water contaminated by faeces of the host infected with C. parvum.4 Numerous outbreaks of cryptosporidiosis are reported on an ongoing basis in both developed and developing countries due to C. parvum contamination of drinking water.5 Considering the lack of curative therapy for cryptosporidiosis,6 it is becoming an urgent issue to monitor quantitative change of C. parvum in water source. So far various methods have been utilized for the detection of C. parvum, such as microscopy,7,8 staining microscopy,9,10 immunofluorescence microscopy detection,11 PCR,12 immune-magnetic separation (IMS) technology,13-18 and electrochemical methods.19,20 Microscopy is the ‘gold standard’ for detecting fecal parasites,21 it is very difficult to distinguish cryptosporidium from other organisms of similar sizes in water samples. Staining microsco-

py such as acid-staining fecal test technique has been used for assay of C. parvum. However, due to the lack of specificity, staining microscopy often results in high false positive rate.10 Immunofluorescence microscopy has been more recently adopted for the identification of C. parvum,11 but the need of fluorescence microscope limits its use for on-site analysis. Conversely, PCR-based methods have been proven to be rapid and sensitive for the detection of C. parvum in lab. However, molecular detection of pathogens remain cumbersome, and there are many species of organisms in natural water, which may leads to false positive results of PCR analysis.22,23 PCR analysis typically requires very clean environment, which limits the application of IMS combined PCR in the context of onsite diagnosis of pathogens. On the other hand, IMS technology has been applied extensively in separating and diagnosing various types of microorganisms, including bacteria,13-15 viruses,16,24 and parasite17,18 from environmental samples due to its prominent advantages such as rapidity, low-cost and effectivity. IMS combined with fluorescent microscropy method25 (named Method 1623, the

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C. parvum detection standard by American EPA) or IMS combined with PCR26,27 both offer an accurate and reliable detection of C. parvum. The limit of detection (LOD) of IMS-coupled quantitative PCR method is 8.7 oocysts. In addition, electrochemical methods show ultrasensitivity by using an electrode containing gold nanoparticle modified with specific antibodies or enzymes.19,20 These methods mentioned above are not available for on-site assay because of the need of strict low background on the operating environment. To date, neither current molecular identification methods nor reported microscopic ones are capable of on-site enumeration of C. parvum in a low-cost, time-effective and facile way. Herein, we propose a robust strategy for on-site counting of C. parvum, which requires only a cheap portable dark-field microscope and a prepared magnetic nanoparticle (MNP) probe.28,29 The scattered golden color of MNP probes under dark-field microscope has been applied previously in biosensing applications.30,31 Our strategy is based on the hypothesis that hundreds of nanometers size of MNP probe can be easily visualized under dark-field microscopy, which allows MNP probecaptured parvum to be easily and selectively visualized under dark-field microscopy in the form of bright garland-like structures (Scheme 1).

taining 0.05% Tween 20, pH = 7.4) containing 1% BSA and stored at 4°C until used. Goat anti-C. parvum polyclonal IgG antibody at 4 mg/mL was added to the washed particles at the final concentration of 10 mg/mL and incubated at room temperature for 60 min in a rotary shaker. The magnetic particle probes were washed three times with PBST to remove the unbound antibodies and collected using magnetic stand (Promega, USA). Then the MNP probes were re-suspended in PBST containing 1% BSA to final concentration of 20 mg/mL and stored at 4°C until use. The concentration of antibodies, before and after magnetic particle probe preparation, was determined by SDS-PAGE assay. Counting of C. parvum by MNP Probes with DarkField Microscope. 10 μL of magnetic nanoparticle probes (10 mg/mL) were added to 10 μL of water samples of C. parvum and 80 μL of PBST. The mixture was incubated at room temperature for 20 min. Then the probes captured C. parvum were collected using magnetic stand. The probes-captured C. parvums were washed three times with PBST, followed by subject to dark-field microscope (Jiangnan Co., China), field emission scanning electron microscopy (FE-SEM; S-4800; Hitachi, Tokyo, Japan) and laser scanning confocal microscope (Leica TCS SP8 STED; Wetzlar, Hessen, Germany) for imaging and enumeration. Quantification of Oocysts by DAPI Stain-Microscopy. C. parvum stock solution (~1.0 × 108 in 8 mL) was serially diluted with PBS into samples at different concentrations. 10 μL of each sample was applied to a clean microscope slide and then was fixed carefully with methanol for 10 min, followed by staining with DAPI for another 10 min. Finally the stained samples were imaged with fluorescent microscope. The whole area of the slide was well screened in a systematic, side-by-side manner × 400 magnification. Numbers of oocytes were counted with the naked eye.

Scheme 1. Schematic diagram of the C. parvum enumeration method based on dark-field microscopy. MNP probes conjugated with anti-C. parvum antibodies were gently mixed with the sample for 20 min after which the probeC. parvum complexes were separated by magnetic force, followed by counting with dark-field microscopy. EXPERIMENTAL METHODS Materials. C. parvum stock solution was purchased from Waterborne™, Inc. (New Orleans, USA). Magnetic particles (~800 nm) were purchased from Creative Diagnostics Inc. (New York, USA). Goat anti-C. parvum polyclonal IgG antibody was purchased from Abcam (Cambridge, UK). Bovine serum albumin (BSA) and 4ʹ, 6-diamidino-2phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PBS was supplied by Biyuntian Co. (Shanghai, China). Methanol and Tween 20 were purchased from Aladdin (Los Angeles, CA, USA). Preparation of Magnetic Nanoparticle Probe. Magnetic particles were prewashed three times with PBST (con-

The Procedure of PCR Analysis. The ability of magnetic particle probes to capture the oocysts of C. parvum was examined by PCR. A fragment of the Cryptosporidium oocyst wall protein (cowp, GenBank Number: Z22537) was amplified with primers (forward primer: 5ʹCAAATTGATACCGTTTGTCCTTCTG-3ʹ; reverse primer: 5ʹ-GGCATGTCGATTCTAATTCAGCT-3ʹ) to detect C. parvum. 1 μL of C. parvum sample solution was used as the template in each of PCR reaction, and PCR was performed according to the following procedure: 1) a predenaturation step at 95°C for 5 min; 2) denaturation at 95°C for 30 s; 3) annealing at 55°C for 30 s; 4) extension at 72°C for 30 min; 5) finally extension at 72°C for 5 min. 30 cycles from 2) to 4) was set up for amplifying the target DNA. The PCR products were then analyzed with 1.5% agarose gel electrophoresis. Acid Disaggregation Assay of Irregular Aggregates. 10 μL of assay mixture containing MNP probes and C. parvum sample was dropped on a clean glass slide and then put on the cover glass. Lenses were adjusted to focus on an irregular aggregate under bright-field condition. Subsequently 5 μL of dilute acid solution was added from one

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ACS Sensors side of the cover glass and the disaggregation process was recorded using the microscope camera. RESULTS AND DISCUSSION Construction of MNP Probe. To demonstrate the aforementioned hypothesis, we first constructed MNP probes by conjugating a ~800 nm spherical MNP precoated with protein G with antibodies specific for C. parvum (Figure S1a (tube 3), S1c and S1e in the Supporting Information). Owing to the characteristic of protein G that had a strong affinity to the Fc region of goatantibodies, the MNP probes could be readily prepared by mixing the protein G functionalized MNP with antibodies, followed by magnetic separation. This MNP probes preparation procedure was straightforward, which was advantageous over the commonly used tedious chemical linker molecular modification methods.32 Electrophoresis result shows the presence of two bands (a ~55 kDa band of antibody heavy chain and a ~20 kDa band of antibody light chain) in the MNP probes (Figure 1), confirming the successful conjugation of the anti-C. parvum antibodies. The binding efficiency, calculated by comparing the gray value of the antibody heavy chain band with those of standard antibody bands using the software Quantity One (Bio-Rad Laboratories, USA), was determined to be approximately 1.5 μg antibody molecules coating on 100 μg of MNP probes (equivalently ~40,000 antibody molecules conjugated on one MNP).

perse by naked eyes (Figure S1a), MNPs and MNP probes could actually form small aggregations of 2 to 3 particles by themselves under microscope (Figure S1b, S1c, S1d and S1e). The resulting solution was further washed three times with PBST, and then subjected to dark-field microscopy. Every oocyst was found to be encircled with numerous MNP probes, which resulted in the formation of dim garland-like structures under bright field microscopy (Figure 2a) and bright yellow garland structures under dark-field microscopy (Figure 2b). This observation was confirmed by SEM (Figure 2c). Furthermore, a red fluorescent MNP modified with protein G was used to support these results. Confocal microscopy confirmed that the MNP probes were present at the surface of C. parvum oocytes and formed garland-like structure (Figure 2d-2f). These structures were significantly different from those associated with excess MNP probes potentially not attached to C. parvum.

Figure 2. Morphologies of MNP probe-conjugated C. parvum visualized by (a) bright field microscopy, (b) darkfield microscopy, (c) SEM, (d) bright field laser confocal microscopy, (e) fluorescent image at 590 nm excitation wavelength of laser confocal microscope and (f) merged image from d and e. The insets in the upper right corner in a or b was the enlargement of the selected areas, respectively.

Figure 1. The combination efficiency of MNP and anti-C. parvum polyclonal antibody. (a) Protein marker. (b) 5 μg anti-C. parvum polyclonal antibody. (c) 2 μg anti-C. parvum polyclonal antibody. (d) 1 μg anti-C. parvum polyclonal antibody. (e) 10 μL of MNP probe. There were approximate 1.5 μg antibody molecules coating on 100 μg of MNP probes by comparing the average gray value of the heavy chain band of 2 μg anti-C. parvum polyclonal antibody with that of 10 μL of MNP probe based on triplicate independent experiments using the software Quantity One. Analysis of Purified C. parvum Samples. Next the prepared MNP probes were employed to capture and count the C. parvum in standard samples under dark-field microscopy. The assay solution containing 10 μL of MNP probes (10 mg/mL, 1.8 × 108 particles), 10 μL of C. parvum (~1.0 × 104) and 80 μL of PBST were gently mixed for 20 min, and then separated using a standard magnet. Although these solution of MNP, MNP probes and MNP probe-conjugated C. parvum appeared to be monodis-

Determination of Optimal Concentration of MNP Probes for the Detection of C. parvum. To determinate the optimal concentration of MNP probes, a certain amount of C. parvum was added to different concentrations of MNP probes solution. 10 μL of C. parvum (~1.0 × 104) was gently mixed with MNP probes at 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL and 4 mg/mL, respectively (figure S2a), and then subjected to dark-field microscopy. Six samples at 0.25-4 mg/mL MNP probes were incubated with C. parvum and imaged under darkfield microscope (Figure 3a-3f). At 1 mg/mL MNP probes, there were more clearly visible garland-like structures (as determined by the number of white arrows in Figure 3), which allowed for reliable detection of the target. Higher concentrations of the MNP probes resulted in more aggregation which in turn reduced the reliability of the method (Figure 3e and 3f). 1 mg/mL was therefore selected as the optimal concentration of MNP.

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Figure 3. Dark-field images of C. parvum captured by different concentration of MNP probes. (a) 0.25 mg/mL, (b) 0.5 mg/mL, (c) 1 mg/mL, (d) 2 mg/mL, (e) 3 mg/mL, (f) 4 mg/mL. The insets in the upper right corner in each graph above are the enlargement of the selected areas. The golden garland-like structure formed by MNP probes and C. parvum were pointed by white arrows. To demonstrate our enumeration method could be employed for the detection of target-of-interest in the real world. MNP probes were mixed with C. parvum oocytes and Salmonella DT104 or E. coli O78, respectively. Results show that MNP probes did not bind with both Salmonella DT104 and E. coli O78 (Figure 4). MNP probes alone also do not bind with Salmonella DT104 or E. coli O78 (Figure S3).

Figure 4. No MNP probes binding with any kind of bacteria when capturing C. parvum oocytes. (a and b) Darkfield images of C. parvum captured by MNP probes in the sample spiking with interference Salmonella DT104 and E.coli O78, respectively. The upper right corner in each graph above was the enlargement of the selected area. (c and d) TEM images of C. parvum captured by MNP probes interference with Salmonella DT104 and E.coli O78, respectively. MNP probes-conjugated C. parvum oocytes were labeled with white arrow, and bacteria with red arrows. Determination of Limit of Detection. A series of samples with different concentrations of C. parvum oocytes in PBS were employed to investigate the LOD, specificity and captured efficiency using the MNP-based dark-field microscopy counting method. Prior to be assessed by our proposed counting method, these C. parvum samples were first treated by DAPI staining so that their number in a specific sample could be confirmed using a standard count plate under a microscope.

These DAPI-stained samples were subjected to the method by counting three samples at different concentrations (500 aM, 50 aM and 5 aM). The counting pictures (Figure 5d-5f) from three samples at 500 aM, 50 aM and 5 aM, were consistent with those obtained by DAPI (Figure 5a5c). At 5 aM concentration, there was occasionally one or two field of views out of ten in which one or two garlandlike structures were still clearly visible (Figure 5f). It was worth mentioning that no false positive results were obtained using the MNP-based counting method because no garland-like structures form in the absence of the target pathogen (Figure S1c). There was no garland-like structures formation when the naked MNPs (without antibody functionalization) were mixed with C. parvum at 500 aM (Figure S4a, S4d, S4g and S4j), 50 aM (Figure S4b, S4e, S4h and S4k) and 5 aM (Figure S4c, S4f, S4i and S4l), respectively. The standard curve was obtained by our counting strategy with six concentration values (500 aM, 200 aM, 100 aM, 50 aM, 20 aM and 5 aM) in table S1. The LOD was determined by the extrapolation of the calibration curve with three times the standard deviation of the background signal (dash black line).33,34 The LOD for dark-field enumeration was equivalent to 5 pathogens in 1 μL (Figure 5g). The sensitivity of the counting strategy was found to be comparable with that of PCR in this study. Indeed, the electrophoresis band of the PCR amplified fragment of cowp gene of C. parvum with sample at 8 aM as template was almost invisible (Figure S5).

Figure 5. Capture efficiency of the MNP probes for the detection of C. parvum. (a, b and c) samples by DAPI staining under a confocal microscope at 500 aM, 50 aM and 5 aM, respectively. (d, e and f) Dark-field images of C. parvum captured by MNP probes in samples corresponding to a, b and c, respectively. (g) The standard curve of the counting strategy according to the concentrations determined by DAPI-stained microscopy and the corresponding values by the MNP counting method (red line). LOD (8 aM) was calculated based on the standard curve. (h and i) Dark-field images of two concentrated real water samples containing C. parvum. The upper right corner in each graph above was the enlargement of the selected area.

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ACS Sensors According to the counted numbers of standard samples (C. parvum in PBS from commercial company) by our MNP-based method and the actual ones determined by DAPI staining and counting method, the calculated capture efficiency values ranged from 50%~80% at 6 different concentrations of C. parvum (Table S1). Apparently, there was higher capture efficiency (78.5%) in the present of target of interest at high concentration (500 aM). As the concentration of the target drops to 5 aM, there was only 50% capture efficiency due to the decrease of collision probability between the MNP probes and the pathogens in a unit time and loosing of some pathogens during pipetting or centrifugation.

Suitability of the Counting Strategy. Considering the inherent limit of the size of single field of view and the near sub-micrometer probes size, it becomes impractical to visualize hundreds of garland-like structures in a field of view. Hence, the MNP-based counting strategy is not suitable for quantifying high concentration of the target pathogens. However, in practice the concentration of C. parvum in waters is usually low, even after standard concentration. Therefore, the proposed MNP-based darkfield microscopy strategy has a high potential for being an efficient method for the enumeration of highly infectious pathogens such as C. parvum in water samples.

Analysis of Real Water Samples Containing C. parvum. To demonstrate the feasibility of the method for the analysis of real water samples contaminated by C. parvum. Three water samples from different lakes were first concentrated by filtration and qualitatively analyzed by PCR, followed by quantifying with our counting strategy. The PCR results verified that two of the three water samples were contaminated with C. parvum (Figure S6, line 3 and 4) and one of them was C. parvum free (Figure S6, line 2). The MNP probes and dark field microscopy not only correctly identified the contaminated samples, but also provided quantitative C. parvum burden as shown in Figure 5h and 5i. The concentrations of C. parvum in these two concentrated samples (10,000-fold enrichment from primitive water) were 220 aM and 174 aM, respectively, based on the calibration curve (Figure 5g, red line). In the real samples analysis using our MNP-based counting strategy, there are several irregular aggregates that might be formed due to nonspecific adsorption in each of views under dark-field microscopy. However we found out that most of those particles with sizes larger than 5 µm in diameter (nine out of ten experiments) were formed from a few of MNP probes surrounding a C. parvum by carefully dropping diluted acid solution to disaggregate them (Figure 6, and the movie in the Supporting Information). But those aggregates with sizes smaller than 5 µm in diameter rarely change and no C. parvum was observed to release from these irregular particles (Figure 6, and SI-movie).

In summary, we have demonstrated a reliable, rapid, userfriendly, and cost-effective approach for on-site quantifying Cryptosporidium oocytes in water samples with a LOD of 8 aM. This MNP probe integrated with dark-field counting strategy possesses significant advantages over standard methods, in particular, rapidity (~30 min) and easy-operability. The method can be easily applied to other water pathogen and has therefore the potential of being a universal tool for on-site quantification of pathogens.

CONCLUSIONS

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. The control experiments for the detection of C. parvum under bright field or dark-field microscope at three different concentrations mixed with unmodified anticryptosporidium antibody MNPs, capture efficiency of MNP probes, PCR analysis of real C. parvum oocytes water samples and the movie about difference of acid disaggregation between aggregates with size greater or less than 5 µm measurements (PDF). SI-movie (ZIP) AUTHOR INFORMATION Corresponding Author *Email: [email protected]

ORCID Benjamin Thierry: 0000-0002-6757-2842 Chih-Tsung Yang: 0000-0003-4878-7589 Vipul Bansal: 0000-0002-3354-4317 Xin Zhou: 0000-0003-2515-704X

Figure 6. Evolution of bright-field screenshots of acid disaggregation process adopted from the attached movie. a, b, c and d were the screenshots from movie at intervals of 2~3 s, respectively. An aggregate with size less than 5 μm was labeled with the virtual frame. An aggregate with size greater than 5 μm was labeled with a solid box. Scale bar: 20 μm.

Author Contributions Xin Zhou conceived the experiments, wrote the manuscript, interpreted the data and supervised the research project. Fenglei Chen and Fang Tang performed the experiments and wrote the manuscript. Chih-Tsung Yang, Xinyao Zhao and Jun Wang performed some of experiments. Chih-Tsung Yang, Benjamin Thierry, Vipul Bansal and Jianjun Dai participated in discussion and revised the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the start-up funds to X. Zhou provided by the Yangzhou University (137011016), a project

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funded by the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD), the Endeavour Research Fellowship by the Commonwealth of Australia, the National Natural Science Foundation of China (31702298) and the Natural Science Foundation of Jiangsu Province, China (BK20140686). V. Bansal acknowleges the Australian Research Council for a Future Fellowship (FT140101285). We thank Prof. Longxian Zhang (Henan Agricultural University) for providing C. parvum. REFERENCES (1) Clark, D. P. New Insights into Human Cryptosporidiosis. Clin. Microbiol Rev. 1999, 12, 554-563. (2) Smith, H. V.; Nichols, R. A. Cryptosporidium: Detection in Water and Food. Exp. Parasitol. 2010, 124, 61-79. (3) Checkley, W.; White, A. C., Jr.; Jaganath, D.; Arrowood, M. J.; Chalmers, R. M.; Chen, X. M.; Fayer, R.; Griffiths, J. K.; Guerrant, R. L.; Hedstrom, L.; Huston, C. D.; Kotloff, K. L.; Kang, G.; Mead, J. R.; Miller, M.; Petri, W. A., Jr.; Priest, J. W.; Roos, D. S.; Striepen, B.; Thompson, R. C.; Ward, H. D.; Van Voorhis, W. A.; Xiao, L.; Zhu, G.; Houpt, E. R. A Review of the Global Burden, Novel Diagnostics, Therapeutics, and Vaccine Targets for Cryptosporidium. Lancet Infect Dis. 2015, 15, 85-94. (4) Current, W. L.; Reese, N. C.; Ernst, J. V.; Bailey, W. S.; Heyman, M. B.; Weinstein, W. M. Human Cryptosporidiosis in Immunocompetent and Immunodeficient Persons. Studies of an Outbreak and Experimental Transmission. N. Engl. J. Med. 1983, 308, 1252-1257. (5) Baldursson, S.; Karanis, P. Waterborne Transmission of Protozoan Parasites: Review of Worldwide Outbreaks - An Update 2004-2010. Water Res. 2011, 45, 6603-6614. (6) Manjunatha, U. H.; Vinayak, S.; Zambriski, J. A.; Chao, A. T.; Sy, T.; Noble, C. G.; Bonamy, G. M. C.; Kondreddi, R. R.; Zou, B.; Gedeck, P.; Brooks, C. F.; Herbert, G. T.; Sateriale, A.; Tandel, J.; Noh, S.; Lakshminarayana, S. B.; Lim, S. H.; Goodman, L. B.; Bodenreider, C.; Feng, G.; Zhang, L.; Blasco, F.; Wagner, J.; Leong, F. J.; Striepen, B.; Diagana, T. T. A Cryptosporidium PI(4)K Inhibitor Is a Drug Candidate For Cryptosporidiosis. Nature 2017, 546, 376-380. (7) Quintero-Betancourt, W.; Gennaccaro, A. L.; Scott, T. M.; Rose, J. B. Assessment of Methods for Detection of Infectious Cryptosporidium Oocysts and Giardia Cysts in Reclaimed Effluents. App.l Environ. Microbiol. 2003, 69, 5380-5388. (8) Ignatius, R.; Klemm, T.; Zander, S.; Gahutu, J. B.; Kimmig, P.; Mockenhaupt, F. P.; Regnath, T. Highly Specific Detection of Cryptosporidium Spp. Oocysts in Human Stool Samples by Undemanding and Inexpensive Phase Contrast Microscopy. Parastol. Res. 2016, 115, 1229-1234. (9) Hohweyer, J.; Dumetre, A.; Aubert, D.; Azas, N.; Villena, I. Tools and Methods for Detecting and Characterizing Giardia, Cryptosporidium, and Toxoplasma Parasites in Marine Mollusks. J. Food Prot. 2013, 76, 1649-1657. (10) Inacio, S. V.; Gomes, J. F.; Oliveira, B. C.; Falcao, A. X.; Suzuki, C. T.; Dos Santos, B. M.; de Aquino, M. C.; de Paula Ribeiro, R. S.; de Assuncao, D. M.; Casemiro, P. A.; Meireles, M. V.; Bresciani, K. D. Validation of a New Technique to Detect Cryptosporidium Spp. Oocysts in Bovine Feces. Prev. Vet. Med. 2016, 134, 1-5. (11) Al-Adhami, B. H.; Nichols, R. A.; Kusel, J. R.; O'Grady, J.; Smith, H. V. Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium Parvum and Cryptosporidium Hominis Oocysts by Immunofluorescence Microscopy. Appl. Environ. Microbiol. 2007, 73, 947-955. (12) Bouzid, M.; Elwin, K.; Nader, J. L.; Chalmers, R. M.; Hunter, P. R.; Tyler, K. M. Novel Real-Time PCR Assays for the

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ACS Sensors tive Detection of Cryptosporidium Parvum Oocysts in Calf Feces. Parasitol. Res. 2014, 113, 2069-2077. (28) Assa, F.; Jafarizadeh-Malmiri, H.; Ajamein, H.; Anarjan, N.; Vaghari, H.; Sayyar, Z.; Berenjian, A. A Biotechnological Perspective on the Application of Iron Oxide Nanoparticles. Nano Res. 2016, 9, 2203-2225. (29) Xie, J.; Chen, K.; Chen, X. Production, Modification and Bio-Applications of Magnetic Nanoparticles Gestated by Magnetotactic Bacteria. Nano Res. 2009, 2, 261-278. (30) Wu, X.; Li, T.; Tao, G.Y.; Lin, R.Y.; Pei, X.J.; Liu, F.; Li, N. A Universal and Enzyme-Free Immunoassay Platform for Biomarker Detection Based on Gold Nanoparticle Enumeration with a Dark-Field Microscope. Analyst 2017, 142, 4201-4205. (31) Li, T.; Xu, X.; Zhang, G.Q.; Lin, R.Y.; Chen, Y.; Li, C.X.; Liu, F.; Li, N. Nonamplification Sandwich Assay Platform for Sensitive Nucleic Acid Detection Based on AuNPs Enumeration with the Dark-Field Microscope. Anal. Chem. 2016, 88, 4188-4191. (32) Lim, M. C.; Lee, G. H.; Huynh, D. T. N.; Hong, C. E.; Park, S. Y.; Jung, J. Y.; Park, C. S.; Ko, S.; Kim, Y. R. Biological Preparation of Highly Effective Immunomagnetic Beads for the Separation, Concentration, and Detection of Pathogenic Bacteria in Milk. Colloids Surf B Biointerfaces 2016, 145, 854-861. (33) Yang, C. T.; Wu, L.; Liu, X.; Tran, N. T.; Bai, P.; Liedberg, B.; Wang, Y.; Thierry, B. Exploiting Surface-Plasmon-Enhanced Light Scattering for the Design of Ultrasensitive Biosensing Modality. Anal. Chem. 2016, 88, 11924-11930. (34) Yang, C. T.; Pourhassan-Moghaddam, M.; Wu, L.; Bai, P.; Thierry, B. Ultrasensitive Detection of Cancer Prognostic miRNA Biomarkers Based on Surface Plasmon Enhanced Light Scattering. ACS sensors 2017, 2, 635-640.

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