Supporting Information

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1 Supporting Information Multiplexed Instrument-Free Bar-Chart SpinChip Integrated with Nanoparticle-Mediated Magnetic Aptasensors for Visual Quantitative Detection of Multiple Pathogens Xiaofeng Wei, Wan Zhou, Sharma T. Sanjay, Jie Zhang, Qijie Jin,, Feng Xu, Delfina C. Dominguez, and XiuJun Li *,,,,# Department of Chemistry and Biochemistry, College of Health Sciences, Biomedical Engineering, Border Biomedical Research Center, and # Environmental Science and Engineering, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States College of Materials Science and Engineering, Nanjing Tech University, Nanjing, , People s Republic of China Bioinspired Engineering and Biomechanics Center, Xi an Jiaotong University, Xi an, , People s Republic of China Corresponding author: XiuJun Li; xli4@utep.edu Reagents and materials. Carboxyl-functionalized iron oxide NPs were obtained from Ocean NanoTech, LLC (Springdale, AR). EDC [1-(3- Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride] was purchased from Advanced Chemtech (Louisville, KY). Sulfo NHS and Perfluoro compound FC-40 (fluorinated oil) were obtained from Thermo Fisher Scientific (Waltham, MA). Chloroplatinic acid, L-Ascorbic Acid, and 30% hydrogen peroxide were bought from Sigma-Aldrich (St. Louis, MO). Besides, some materials including Polymethyl Methacrylate (PMMA) sheets with 2 mm thickness, circular magnets, clamps, quadrate rubber, and aluminum sheets were purchased from McMaster-Carr Supply Company (Los Angeles, CA). The food dye and apple juice were purchased from Walmart (El Paso, TX). The apple juice was added with 10 PBS to maintain a similar ph and ionic strength as 1 PBS buffer before it was introduced to the MB-SpinChip. All other chemicals were purchased from Sigma (St. Louis, MO) and were used without further purification unless otherwise noted. Ultrapure Milli-Q water (18.2 MΩ.cm) from a Milli- Q water system (Bedford, MA) was applied to make aqueous solutions. Bacterial Pathogen Culture. The S. enterica (ATCC 14028), E. coli (ATCC 25922) and L. monocytogenes (ATCC 15313) were obtained from American Type Culture Collection (ATCC, Rockville, MD). These three kinds of bacterial pathogens were grown in TSA II agar plates supplemented with 5% sheep blood (Becton Dickinson, Sparks, MD). All the microorganisms were incubated at 35 C for 24 h in an aerobic environment. Different dilutions of bacterial samples were prepared with 10 mm PBS (ph 7.4) or pretreated apple juice. Colonies on plates were counted to determine the number of colony-forming units per milliliter (CFU/mL). Preparation of the DNA Biosensor. The DNA biosensor was composed of two functional DNA probes, beads-dna for the immobilization of DNA biosensor and PtNPs-aptamer for the specific pathogen recognition and nanoparticle-mediated pressure amplification. First, cdna-nh 2 containing the same DNA sequence as the aptamer was conjugated with the carboxyl magnetic beads (beads-cooh). 1 ml 0.25 mg/ml of carboxyl beads were conjugated with 200 nm cdna-nh 2 through the crosslink of 12.5 µg/ml EDC HCl and 15 µg/ml Sulfo- NHS. The mixture solution was adjusted to ph 8.0 by NaHCO 3 and kept shaking for 3 hours at room temperature. The conjugated beads-dna was centrifuged (10000 rpm, 5 min), washed with a 1 PBS buffer for 3 times, and finally solved in 1 PBS as the 100 1

2 nm stock solution of beads-dna. For the PtNPs-aptamer, the aptamer was synthesized with thiol-modification for the chemical conjugation with the PtNPs (See Figure S-1) which were synthesized according to the protocol from a previously published report. 1 TEM was used to characterize their morphology using a Transmission Electron Microscope (TEM) from JEOL Ltd (Peabody, MA). 1 µm thiol-aptamer (50 µl) was activated with 0.1 mm Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) by shaking 1 hour at room temperature. The prepared fresh PtNPs (200 µl, 25 nm) and 2 PBS buffer (250 µl) was added into the activated thiolaptamer solution and kept shaking for 24 hours in darkness. The conjugated PtNPs-aptamer was centrifuged at rpm for 5 min, then washed with 1 PBS buffer for 3 times, and finally dissolved in 1 PBS (500 µl) as the 100 nm stock solution of PtNPsaptamer. Further, 500 µl 100 nm of beads-dna and the corresponding PtNPs-aptamer (100 nm) were mixed and centrifuged (10000 rpm, 5 min) to remove the supernatant, and finally dissolved in a 1 ml binding buffer. The hybridization reaction was incubated at 37 for 4 hours to obtain the DNA biosensor. The hybridized DNA biosensor was centrifuged at rpm for 5 min, and washed with 1 PBS buffer for 3 times, before it was dissolved in 1 PBS (500 µl) as the 100 nm stock solution of the DNA biosensor. Assay Procedure on the MB-SpinChip. Different concentrations of samples were prepared from 10 ~ 800 CFU/mL for S. enterica, 10 2 ~ 10 8 CFU/mL for E. coli, 10 2 ~ 10 7 CFU/mL for L. monocytogenes with the final volume of 40 µl. First, the top Layer-1 was spun to locate the exhaust outlets of Layer-1 and Layer-2 and connect the four branched channels of Layer-1 to four sample inlets of Layer-2 (See Figure S-3b). The sample solution was injected from the inlet of Layer-1 and distributed into four sample recognition microwells to react with the preloaded DNA biosensor. Then, the top Layer-1 was spun with a certain angle to seal all sample inlets and exhaust outlets and formed a hermetic reaction chamber by the clamp (See Figure S-3c). The sample was incubated with DNA biosensors for the specific recognizing reaction for 10 min at room temperature. Through the direction from the sample recognition microwell to the amplification microwell, the sample solution including the PtNPs reporter was shaken into the amplification microwell and mixed with the preloaded H 2 O 2 (See Figure S-3d). A moderate oxygen-producing reaction was initialized by the PtNPs catalyst in the presence of H 2 O 2 and increased the pressure of the sealed reaction chamber. Under the increasing pressure, the food dye was pushed into the bar-chart channel and generated a visual bar chart with a quantitative length. Finally, the mean length of four bar charts after 10 min reaction was calculated and recorded as the readout result. 2

3 Table S-1. Sequences of pathogen aptamers DNA name Sequence (5-3 ) S. enterica aptamer 2 SH-TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G S. enterica cdna NH 2 -CTG TCA TAA TGT CAA GTC CCC GTC GGG TGA CGC CGC CAT A E. coli aptamer 3 SH-CCG GAC GCT TAT GCC TTG CCA TCT ACA GAG CAG GTG TGA CGG E. coli cdna NH 2 -CCG TCA CAC CTG CTC TGT AGA TGG CAA GGC ATA AGC GTC CGG L. monocytogenes aptamer 4 SH-TAC TAT CGC GGA GAC AGC GCG GGA GGC ACC GGG GA L. monocytogenes aptamer NH 2 -TCC CCG GTG CCT CCC GCG CTG TCT CCG CGA TAG TA 3

4 Table S-2. Response of MB-SpinChip for pathogens in the single detection, the multiplexed detection and the detection of the spiked samples Single detection* S. enterica Mean SD RSD E. coli Mean SD RSD L. mono Mean SD RSD 0 CFU/mL % 0 CFU/mL % 0 CFU/mL % 10 CFU/mL % 10² CFU/mL % 10² CFU/mL % 50 CFU/mL % 10³ CFU/mL % 10³ CFU/mL % 100 CFU/mL % 10⁴ CFU/mL % 10⁴ CFU/mL % 200 CFU/mL % 10⁵ CFU/mL % 10⁵ CFU/mL % 400 CFU/mL % 10⁶ CFU/mL % 10⁶ CFU/mL % 600 CFU/mL % 10⁷ CFU/mL % 10⁷ CFU/mL % 800 CFU/mL % 10⁸ CFU/mL % Multiplexed detection** S. enterica Mean SD RSD E. coli Mean SD RSD L. mono Mean SD RSD 0 CFU/mL % 0 CFU/mL % 0 CFU/mL % 200 CFU/mL % 10⁵ CFU/mL % 10⁵ CFU/mL % Detection of the spiked samples** S. enterica Mean SD RSD E. coli Mean SD RSD L. mono Mean SD RSD 0 CFU/mL % 0 CFU/mL % 0 CFU/mL % 50 CFU/mL % 10³ CFU/mL % 10³ CFU/mL % 100 CFU/mL % 10⁴ CFU/mL % 10⁴ CFU/mL % Mean value, standard deviation (SD) and relative standard deviation (RSD) were obtained from four parallel measurements. * Intra-assay: parallel results from one MB-SpinChip ** Inter-assay: parallel results from four MB-SpinChips 4

5 Figure S-1. TEM photograph of PtNPs 5

6 Figure S-2. Schematic of the MB-SpinChip. (A) Layout design of different layers. (B) 3D schematic of the Spin-unit section. 6

7 Figure S-3. Operation steps of the MB-SpinChip, including (a) Standby condition: preloading H 2 O 2 and Food dye, (b) Connect and inject: opening exhaust outlet and connecting the branched channels with sample inlets for sample injection, (c) Spin and seal: spinning the Spin-unit to seal the exhaust outlets and sample inlets, (d) Shake and read: shaking down the samples into the amplification microwells to activate the O 2 generation with the bar-chart readout. 7

8 Figure S-4. Selectivity investigation of the MB-SpinChip with different DNA probes for (A) S. enterica, (B) E. coli, and (C) L. monocytogenes. The standard deviation was obtained from four parallel measurements. 8

9 Figure S-5. Response of MB-SpinChip operated by multiple users to 20 pm PtNPs and 30% H 2 O 2 in 5 min. The relative standard deviations were obtained from four parallel measurements in one assay. 9

10 (c) (a) Preload the reagents (b) (d) Cover the Spin-unit (g) Shake down (f) Spin the (e) the sample Spin-unit Inject the sample Read the bar-length (h) The upper side Connect the branched channels with the sample inlets Seal the exhaust outlets and the sample inlets Anticlockwise spin to align the upper side The bottom side Figure S-6. Different construction stages of the MB-SpinChip by the assay procedure, including (a) prepared Reaction-unit, (b) preloading H 2 O 2 (light blue) and food dye (yellow, dark blue, red and green circle) into the Reaction-unit, (c) prepared Spin-unit (light orange), (d) covering the Spin-unit to align the bottom side of the reaction-unit, (e) injecting the sample (light green) via the Spin-unit, (f) spinning the Spin-unit to seal all the microwells, (g) shaking the samples into the amplification microwells, and (h) reading the bar-length signal. Besides, the spin operating from (e) to (d) is partly enlarged in the illustration. 10

11 References (1) Xie, Y.; Wei, X.; Yang, Q.; Guan, Z.; Liu, D.; Liu, X.; Zhou, L.; Zhu, Z.; Lin, Z.; Yang, C. Chem. Commun. 2016, 52, (2) Zuo, P.; Li, X.; Dominguez, D. C.; Ye, B. C. Lab Chip 2013, 13, (3) Wu, W.; Zhang, J.; Zheng, M.; Zhong, Y.; Yang, J.; Zhao, Y.; Wu, W.; Ye, W.; Wen, J.; Wang, Q. PloS one 2012, 7, e (4) Duan, N.; Ding, X.; He, L.; Wu, S.; Wei, Y.; Wang, Z. Food Control 2013, 33,