16S rRNA Monitoring Point-of-Care Magnetic Focus Lateral Flow Sensor.

The detection and profiling of pathogenic bacteria is critical for human health, environmental, and food safety monitoring. Herein, we propose a highly sensitive colorimetric strategy for naked eye screening of 16S ribosomal RNA (16S rRNA) from pathogenic agents relevant to infections, human health, and food safety monitoring with a magnetic focus lateral flow sensor (mLFS) platform. The method developed was demonstrated in model 16S rRNA sequences of the pathogen Escherichia coli O157:H7 to detect as low as 1 fM of targets, exhibiting a sensitivity improved by ∼5 × 105 times compared to the conventional GNP-based colorimetric lateral flow assay used for oligonucleotide testing. Based on the grayscale values, semi-quantitation of up to 1 pM of target sequences was possible in ∼45 min. The methodology could detect the target 16S rRNA from as low as 32 pg/mL of total RNA extracted from pathogens. Specificity was demonstrated with total RNA extracted from E. coli K-12 MG1655, Bacillus subtilis (B. subtilis), and Pseudomonas aeruginosa (P. aeruginosa). No signal was observed from as high as 320 pg/mL of total RNA from the nontarget bacteria. The recognition of target 16S rRNA from 32 pg/mL of total RNA in complex matrices was also demonstrated. The proposed mLFS method was then extended to monitoring B. subtilis and P. aeruginosa. Our approach highlights the possibility of extending this concept to screen specific nucleic acid sequences for the monitoring of infectious pathogens or microbiome implicated in a range of diseases including cancer.

Introduction

Pathogens induce various health threats from foodborne diseases to infection-related illnesses. The increasing demand for rapid screening methods has drawn extensive attention from researchers and consequently various approaches have been proposed. Methods have been developed to detect specific surface antigens of pathogens based on antibody recognition methods.[1−8] Disadvantages of the antibody–antigen interaction-based techniques are the following: (i) potential cross-reactivity resulting in false positives;[9−11] (ii) low expression of indicator antigens on the surface of the target organism, resulting in unsatisfactory sensitivity and inconsistent results; and (iii) possible denaturation of antibodies, resulting in the loss of recognition specificity. An alternative to antigen monitoring could be based on nucleic acid sequence detection, for example, 16S rRNA could serve as an excellent indicator of infectious agents. 16S rRNA has been used to differentiate the target from non-target bacteria with amplification techniques such as polymerase chain reaction or loop-mediated isothermal amplification.[12−14] Amplification-based approaches are complex for onsite screening; hence, colorimetric methods could serve as alternatives.

The lateral flow assay (LFA) is a rapid and simple analytical strategy used predominantly in point-of-care applications and onsite monitoring. The predominant mode of detection with LFA is based on colorimetric signals on paper strips requiring no extra instrumentation and is achievable at a very low cost. LFA has been utilized in a range of applications for bacteria detection,[15−20] including the detection of nucleic acid sequences.[21−25] Although LFA is an attractive analytical tool, its application is limited due to poor sensitivity. Nanoparticles with a high extinction coefficient are commonly used for naked-eye determination in LFA devices.[26,27] A further enhancement of the signal with silver staining or enzyme-based amplification has been proposed to improve detection performance.[1,16,28,29] Besides signal enhancement, another route to improve signal generation is to increase the capture efficiency of labeled targets on the lateral flow (LF) strips by increasing the interaction time with antibodies at the signal generation zone by slowing the mobility of targets as they move through the lateral flow strip. It has been stated that less than 1% of targets are captured on the strip in conventional LFA.[30] Strategies reported to increase the capture efficiency by increasing the reaction time between the targets and the capture ligands conjugated on the LF strip include the concept based on wax pillars printed on the LF strip to generate various patterns which could induce a turbulent flow, and longer reaction times for the capture of targets, resulting in a slightly improved sensitivity.[31] With a sponge attached between the conjugate pad and the nitrocellulose (NC) membrane, the obtained LF strip could reduce the flow rate and enable a 10-time enhanced sensitivity.[32] To optimize the movement of the probes, an electrophoresis approach, isotachophoresis (ITP), was utilized with LFA.[30,33,34] The charged probes would be controlled by the voltage applied on the LF strip. Although an improvement in sensitivity could be achieved with the ITP-LFA strategy, additional instruments and test setups were required.

Previously, an improved LF biosensor termed as the magnetic focus enhanced lateral flow assay (mLFA) was proposed to improve the capture efficiency of targets on the LF strip for enhanced sensitivity. To control the movement and distribution of labeled probes on targets in the sample flow on the LF strip, Fe3O4/Au core–shell magnetic nanoparticles (mNPs) were used to develop the probes. A simple external magnet was placed under the test zone of the LF strip to tune the flow speed and distribution of the magnetic probe-labeled targets on the strip. The longer reaction times and higher concentrations of the labeled target in the region near to the antibody conjugated in the test zone resulted in a higher capture efficiency. Benefiting from the horseradish peroxidase (HRP)-based amplification of the colorimetric signal, the mLFA exhibited greatly improved sensitivity, approximately 106 times higher than conventional LFA for the protein biomarker detection and more than 400 times higher for detecting bacterial cells.[35,36]

Although the detection of 16S rRNA could be achieved with the enhancement of nucleic acid amplification, the requirement from the amplification step limits the application of the corresponding biosensors. Herein, an mLFS test for 16S rRNA was proposed based on a colorimetric signal without any RNA amplification. Escherichia coli O157:H7 is a Shiga toxin-producing bacterium that can induce illnesses ranging from mild diarrhea to fatal hemorrhagic colitis accompanied by bloody diarrhea and thrombotic thrombocytopenic purpura with as high as 40% death rates.[37−39] The proposed mLFS could recognize as low as 1 fM of model DNA sequences of a segment of 16S rRNA from E. coli O157:H7 by the naked eye, indicating an improvement in sensitivity by 5 × 105 times than that from the GNP-based colorimetric LFA method.[40]

Further tests were performed to show that the mLFS method could detect the presence of 16S rRNA in as low as 32 pg/mL of total RNA extracts from E. coli O157:H7. Specificity of the mLFS was further tested with the total RNA extracted from E. coli K-12 MG1655, Bacillus subtilis, and Pseudomonas aeruginosa. The testing of select organisms, E. coli O157:H7, in complex mixtures (e.g., in lettuce) was demonstrated to illustrate the range of applicability. With the corresponding probe sequences (PSs) and capture sequences (CSs) designed for different pathogens, the proposed mLFS strategy was also used to detect B. subtilis and P. aeruginosa, demonstrating its ability to monitor multiple organisms. The sensitivity and specificity achieved in detecting RNA in complex matrices opens a range of possibilities in health screening, microbiome monitoring, and food safety applications.

Results and Discussion

The detection of nucleic acid sequences, as shown in Scheme , consists of the CS/target sequence (TS)/PS sandwich structure, where the CSs were labeled with specific chemicals for capture by the antibody on the LF strip. The sandwich structure links the labels to the TSs with good specificity.[41,42] The capture of labels on the CS/TS/PS-GNP sandwich structures would link the GNP probes to the LF strip with the color from GNPs in the presence of TS. Driven by the sample flow, the short interaction time between the labels on the CS/TS/PS-GNP sandwich structures and the antibodies conjugated on the LF strip would result in a low capture efficiency and a very limited number of GNP probes linked to the LF strip; thus, a low detection sensitivity at the sub-nM level could be achieved. In contrast, in mLFS, HRP was linked to the magnetic probes to catalyze the tetramethyl benzidine (TMB)-based colorimetric reaction. Although the HRP on the unbound magnetic probes would generate a green color beyond the test zone on the LF strip, the higher concentration of probes immobilized in the test zone due to the presence of TSs would induce observable green spots in the test. Thus, green spots were used to determine the existence of TSs. With the obtained green product, the colorimetric signal can be greatly amplified. More importantly, the magnetic focus effect due to the magnetic force between the magnetic probe and the magnet under LF strips would tune the movement and distribution of the labeled CS/TS/PS-magnetic probes in the sandwich structures. From our previous work, the magnetic focus effect would slow down the rate of movement of the magnetic probes by ∼10% compared to that without magnetic focus, and the magnetic probes would be concentrated to the walls of the microchannels in the LF strip where the antibodies are conjugated.[35] Compared to the 0.5 nM sensitivity obtained from the GNP-based colorimetric LFA with a similar recognition sandwich pattern labeled CS/TS/PS-NPs in a 15 min detection procedure,[40] the detection time of the proposed mLFS is 45 min with the ability to recognize as low as 1 fM of the target nucleic acid sequences at a comparable cost.

Scheme 1

Comparison between GNP-Based Colorimetric LFA and the Proposed mLFS Platform for the Detection of 16S rRNA

In this work, when the mLFS method was applied for RNA detection, the magnetic focus would tune the labeled CS/TS/PS-magnetic probe sandwich structures in the sample flow, moving it closer to the antibody on the wall of the microchannels at a slower rate. The resulting longer reaction time and higher concentration of labels on the sandwich structures would increase the capture efficiency to result in better sensitivity. Although the competition with unreacted labeled CS remains, the magnetic focus would improve the detection sensitivity of the target nucleic acid sequences.

The mNPs were synthesized based on our previous works, which were nanoparticles around 60–80 nm.[35,36] To characterize the preparation of the mNPs, as shown in Figure A, UV–vis spectra are recorded after each step of the modification. The extinction of nanoparticles is affected by the surface plasmon resonance (SPR). During probe preparation, the conjugation of DNA sequences, casein, and HRP on the surface of mNPs would influence the SPR, which could be revealed by the peak shift and absorption change in the UV–vis spectra. In Figure A, it can be seen that there is a slight red shift upon SH-PS conjugation, while the increase in absorption in the long wavelength range suggests the aggregation of mNPs during the PS conjugation. The casein block induced a clear red shift of the mNP peak, implying that a shell of casein has formed around mNPs. A slight red shift due to the conjugation of SA-HRP was observed, which should be attributed to the HRP molecules immobilized on the casein layer far from the surface of mNPs with a smaller influence due to SPR. Meanwhile, the obvious increase in the absorption at the longer wavelength range should be attributed to the multiple binding sites on the streptavidin of the SA-HRP cross-linked magnetic probes. Furthermore, by measuring the concentration of PS before and after the conjugate, the amount of SH-PS on each mNPs was calculated. The concentration of mNPs, which were wrapped in gold shells, was determined based on the reported method.[43] Based on the concentration change of PS upon the modification and concentration of mNPs, the average number of PS per mNP was calculated to be approximately 4517. To further confirm the modification of probes, zeta potential measurements of the mNPs, PS-mNPs, probes, and probes conjugated with HRP were performed, and the results are shown in Table . The change in the zeta potential upon immobilization of PS, blocking with casein, and conjugation of HRP could be noted. The morphology of the mNPs was characterized by transmission electron microscopy (TEM), which is shown in Figure B. It can be seen that the mNPs exhibited nonuniform shapes at the size from around 50 to 100 nm. The magnetic response of the mNPs was demonstrated with an external magnet and the concentration of the mNPs is shown in Figure C. It can be seen that after 3 min, most of the mNPs were concentrated to the side where the magnet was placed. After 10 min, all the mNPs were concentrated at the tube wall close to the magnet.

Figure 1

UV–vis spectra of mNPs, SH-PS-modified mNPs, casein-blocked SH-PS-modified mNPs, and magnetic probe immobilized with HRP (A). TEM image of mNPs (B) and its response to a magnet (C) where 0 min corresponds to the mNPs after sonication.

Table 1

Zeta Potential of Nanostructures upon Modification

	mNPs	PS-mNPs	probes	HRP-probes
zeta potential/mV	–23.0	–24.2	–20.3	–18.3

To achieve the best detection sensitivity, the concentration of HRP-probes was optimized, and the results are shown in Figure S1. Under the optimized detection conditions, the TS at serial concentrations was detected with the proposed mLFS method. A typical image of the strip along with the quantitative results is shown in Figure . It can be observed that with 1 fM of target TS a spot appeared on the LF strip as the signal to determine the presence of TSs. In contrast, no spot was observed on the LF strip where blank samples were tested, although a light green color was noted all over the NC membrane in the strips which should be attributed to the HRP on the unbound magnetic probes. Therefore, in the proposed mLFS, the determination of the existence of TSs was based on the appearance of green spots, while the semi-quantitation of the concentration of the TSs was based on the grayscale of the color in the test zone. It can be seen from the image that the color of the spots became deeper with higher concentrations of the target TS. The quantitative results correspondingly showed larger grayscales with an increase in the concentration of TS. In the quantitative results, a deviation of the grayscale from blank samples was noted which was assigned to the change in color all over the strips with no spots, which should be attributed to the varying amount of unreacted probes trapped in the NC membrane. In the mLFS, the magnetic focus improved the capture efficiency, meanwhile, the slower movement and concentrated distribution of magnetic probes would keep more probes with HRP on the strips even after the cross-flow wash, resulting in a grayscale value from the blank samples.

Figure 2

Detection results for TS at serial concentrations and the corresponding quantitative grayscale results.

After the validation of the proposed mLFS for the detection of short oligonucleotide model TS, 16S rRNA in the total RNA extracted from different pathogens was tested. To retain the simplicity of the LF platform-based method, no isolation of 16S rRNA from the total RNA extracted from E. coli O157:H7 was performed to demonstrate the recognition capability of the mLFS platform. Due to the secondary structure of 16S rRNA, FITC-CS was added to the sample solution along with the magnetic probes, and the incubation time increased to 15 min. Considering the interaction between FITC-CS and magnetic probes, and thereby, the signal from blank samples, the amount of FITC-CS used in the experiment is investigated, and the results from the varying amounts of FITC–FITC are shown in Figure . It can be seen that with 1 μL of 1 pM FITC-CS, no spot was observed from as high as 320 pg/mL of the total RNA and an unclear dot appeared from the 3.2 ng/mL samples. When the concentration of FITC-CS increased to 10 pM, a dot was visible from 32 pg/mL of total RNA; meanwhile under the same detection conditions, no signal was noted from the blank samples. With 100 pM of FITC-CS, the color of the spots from all of the tested concentrations was deeper than that with 10 pM FITC-CS; however, spots from the blank samples were also observed. Therefore, for the best detection performance, 10 pM of FITC-CS is an optimal concentration. It can be seen that with 1 μL of 10 pM FITC-CS, 16S rRNA in 32 pg/mL of total RNA could be recognized with mLFS, while the color of the spot deepened with higher concentrations of total RNA. The results shown in Figure suggest that the FITC-CS would benefit from the recognition of the target section of 16S rRNA, but the nonspecific interaction between FITC-CS and magnetic probes would result in spots from the blank samples and influence the determination of 16S rRNA.

Figure 3

Detection results for 16S rRNA in serial dilutions of total RNA extracted from E. coli O157:H7 at different levels of FITC-CS.

To characterize the specificity of the proposed mLFS method for 16S rRNA detection from E. coli O157:H7, total RNA from three bacteria, E. coli K-12 MG1655, B subtilis, and P. aeruginosa, are extracted and tested under the same experimental conditions, and the obtained results are shown in Figure . The concentration of the total RNA was diluted to be the same as that from E. coli O157:H7. With 3.2 and 320 ng/mL of the total RNA from the three organisms, no spot was observed in the test zone, demonstrating the excellent specificity of the mLFS platform for 16S rRNA detection. Because the detection of target 16S rRNA is based on sandwich structures generated from selective hybridization between the PS sequences on probes, target 16S rRNA and CS-FITC, the change in the zeta potential of the HRP-probes modified with PS or BSPS mixed with the TS sequences and FITC-CS for the 16S rRNA from E. coli O157:H7 is presented in Table . It can be seen that the zeta potential of the HRP-probes modified with PS changed from −18.3 to −19.6 mV after mixing with the TS sequences and FITC-CS, demonstrating the hybridization and generation of sandwich structures. In contrast, the zeta potential of the HRP-probes modified with BSPS remained the same after mixing with the TS sequences and FITC-CS, indicating the specificity of the mLFS method due to hybridization between the target 16S rRNA and the corresponding PS and CS sequences.

Figure 4

Specificity results with RNA extracted from E. coli K-12 MG1655, B. subtilis, and P. aeruginosa.

Table 2

Zeta Potential Change upon the Formation of a Sandwich Structure

	E. coli O157:H7 probes with HRP	E. coli O157:H7 probes with HRP + E. coli O157:H7 TS and FITC-CS	BS probes with HRP	BS probes with HRP + E. coli O157:H7 TS and FITC-CS
zeta potential/mV	–18.3	–19.6	–17.3	–17.3

To demonstrate the applicability of the technology in complex matrices, an example of detecting 16S rRNA extracted from E. coli O157:H7 diluted in the processed lettuce solution at serial concentrations is shown in Figure . An unclear spot could be seen from the 32 pg/mL of total RNA in the lettuce solution, and the spot was more pronounced at 320 pg/mL or a higher concentration of total RNA. Compared to the detection of total RNA diluted in PBS buffer, the color of the spots from the total RNA diluted in lettuce was lighter, suggesting an influence in the formation of the sandwich structure of the FITC-CS/target 16S rRNA/PS-magnetic probe and the capture of the FITC label conjugates by the antibodies immobilized on the LF strip as expected. The results indicate that the technology can be extended to monitoring a range of food and environmental samples.

Figure 5

Detection results for serial concentrations of RNA extracted from E. coli O157:H7 diluted in the lettuce solution.

To demonstrate the potential for detecting various pathogens based on 16S rRNA, probes were prepared with SH-BSPS designed for the 16S rRNA from B. subtilis and with SH-PAPS for the 16S rRNA from P. aeruginosa. The mLFS method was shown to detect B. subtilis with SH-BSPS and BSCS-FITC, and P. aeruginosa with SH-PAPS and PACS-FITC with the same protocol used for the E. coli O157:H7 tests shown above. The results from the detection in the total RNA extracted from B. subtilis and P. aeruginosa are presented in Figure . It can be seen that for both B. subtilis and P. aeruginosa, the target 16S rRNA could be recognized from as low as 320 and 32 pg/mL of the total RNA extracts, respectively; meanwhile, the color of the spots on the LF strip became more pronounced with higher concentrations of total RNA, implying the potential for quantification by colorimetry. The detection results from B. subtilis and P. aeruginosa clearly demonstrate the broad applicability of the mLFS sensor developed for 16S rRNA screening and, in the long-term, for microbiome profiling.

Figure 6

mLFS detection of B. subtilis with probes modified with SH-BSPS and BSCS-FITC and P. aeruginosa with probes modified with SH-PAPS and PACS-FITC.

Conclusions

A highly sensitive 16S rRNA screening strategy was developed based on a mLFS platform. The method developed was simple and practical, yielding excellent sensitivity. As low as 1 fM of model DNA oligonucleotides could be recognized by the naked eye, and the target 16S rRNA could be recognized from as low as 32 pg/mL of the total RNA extracted from E. coli O157:H7 in PBS buffer as well as in the processed food matrix. The proposed mLFS method was then extended to monitor B. subtilis and P. aeruginosa by detecting 16S rRNA extracted from these organisms to show the broader applicability when testing for infections and contamination of environmental and food samples. The enhanced detection sensitivity was achieved without extra instruments or complex steps, thus retaining the simplicity and practicality of the mLFS system with a high degree of specificity. In the long-term, our approach could serve as a robust platform for monitoring infectious pathogens in human, environmental, and food samples.

Experimental Methods

Chemicals and Agents

FeCl3, FeCl2, HAuCl4·XH2O, sodium citrate, sodium carbonate, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma (MO, US). NaOH was obtained from Mallinckrodt Chemicals (NJ, US). NaBH4 was ordered from ACROS ORGANICS (NJ, US). The tetramethyl benzidine (TMB) substrate solution was purchased from Moss Inc. (MD US). Luria Bertani (LB) broth, Sulfo-NHS-LC-Biotin, and streptavidin poly-horseradish peroxidase (SA-HRP, catalog number: 21130) were obtained from Thermo Fisher Scientific (NY, US). Diethyl pyrocarbonate (DEPC)-treated water was purchased from Invitrogen (CA, US). DNA sequences for capturing sequence (CS), probing sequence (PS), and target sequence (TS) were purchased from IDT DNA Technology (IA, US). Sequences designed are as follows: for E. coli O157:H7, CS-FITC: GGC CAA TGT TTG TAA TCA GTT CCT TTT TTT TTT/36-FAM/; SH-PS:/5ThioMC6-D/TT TTT TTT TTT CCA TGC CAA TGC GCG ACA T; TS: GGA ACT GAT TAC AAA CAT TGG CCG CAA ATT GCA CAA TTT GCC CTC GGA ATG TCG CGC ATT GGC ATG GA; for B. subtilis, BSCS-FITC: TGT CTC AGT CCC AGT GTG GTT TTT TTT TT/36-FAM/; SH-BSPS:/5ThioMC6-D/TTT TTT TTT TCG TAG GAG TCT GGG CCG; for P. aeruginosa, PACS-FITC: CTT TGT ACC GAC CAT TGT AGC TTT TTT TTT T/36-FAM/: SH-PAPS:/5ThioMC6-D/TTT TTT TTT TCA CCT CGC GGC TTG GCA ACC. Anti-fluorescein isothiocyanate (FITC) antibody (ABIN6391433) was obtained from antibodies-online Inc. (PA, US). E. coli O157:H7 was acquired from Dr. Feng Hao (Food and Bioprocess Engineering), B. subtilis 168 from Dr. James Imlay (Department of Microbiology), and P. aeruginosa PA14 from Dr. Rachel Whitaker (Department of Microbiology) at the University of Illinois at Urbana-Champaign (UIUC) in the current study. All chemicals and reagents were used as received without further purification; all glassware was cleaned with fresh aqua regia and then washed with deionized (DI) water six times before use.

Bacteria Culture and RNA Extraction

Bacterial strains were grown in the LB broth at 37 °C for 8 h and 1 mL of each culture was serially diluted and plated on the LB agar to count the colony forming units. 1 mL from the same bacterial broth cultures were processed to isolate the total RNA by the Trizol method (Ambion, Thermo Fisher, MA, US). The isolated RNA was dissolved in 40 μL of DEPC-treated water. The concentration and purity of the isolated RNA were analyzed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., MA, US).

Preparation of Magnetic Fe3O4/Au Core–shell Nanoparticles

Fe3O4 nanoparticles were synthesized based on the reported protocols.[44,45] Briefly, 3 mL of 1 M NaOH was added to 27 mL of DI water, the resulting solution was heated to boiling. Then, 2 mL of 0.4 M sodium citrate was injected. Under strong stirring, 1 mL of 0.2 M FeCl2 and 1 mL of 0.4 M FeCl3 were simultaneously added to the obtained solution, while the color of the solution changed quickly from colorless to black. The solution was then refluxed for 4 h, and the resulting Fe3O4 nanoparticles were then washed with ethanol and water three times, respectively, and then dispersed in 10 ml of DI water.

The preparation of Fe3O4/Au core–shell nanoparticles was carried out based on our previous works.[35,36] Typically, 80 μL of Fe3O4 was dissolved in 920 μL of DI water, and the obtained solution was sonicated for 10 min and then centrifuged at 500 rpm for 10 min. To the supernatant, 100 μL of 1% (w/v) HAuCl4 was added, followed by 10 min of sonication. Then, 200 μL of 10 mM fresh and ice-cold NaBH4 was quickly added, and the obtained solution was sonicated for 10 min. The resulting Fe3O4/Au core–shell mNPs were washed with DI water three times and kept at 4 °C until use. The Au shell of the mNPs facilitated the modification of the mNPs with PS sequences and protein blocking and provided good biocompatibility. TEM images were recorded with a FEI Tecnai G2 20.

Modification of mNPs

The conjugation of SH-PS to Fe3O4/Au core–shell nanoparticles was performed based on our prior work with minor modifications.[46] Briefly, in 485 μL of DI water, 5 μL of 1 mM TCEP, and 10 μL of 100 μM of SH-PS was mixed well, and the obtained solution was kept at room temperature for 1 h. Then, 500 μL of mNPs was centrifuged, and the supernatant was removed. The pellet of mNPs was then redispersed in the solution with the TCEP-treated SH-PS, which was then kept at room temperature for 48 h. The modified mNPs were washed with DI water three times and redispersed in 500 μL of DI water.

The immobilization of HRP was conducted based on our published work with minor modifications. To 500 μL of DNA-modified mNPs, 2 μL of 0.5 M Na2CO3 and 50 μL of 10 mM PB buffer were added, and the resulting solution was shaken for 10 h. Then, 55 μL of 5% (w/v) casein in 10 mM PBS was added to block the SH-PS-modified mNPs, and the obtained solution was shaken for 24 h. The casein-blocked mNPs were washed with 10 mM PBS buffer 3 times and redispersed in 500 μL of 10 mM PBS. To the obtained solution, 10 μL of 1 μg/mL Sulfo-NHS-LC-Biotin in 10 mM PBS was added. The solution was then shaken for 1 h at room temperature, followed by the addition of 50 μL of 5% (w/v) casein and shaken for 1 h. The obtained nanoparticles were centrifuged and washed with 200 μL of PBS buffer 2 times and redispersed in 200 μL of 0.5% (w/v) casein in 10 mM PBS. To conjugate HRP to mNPs, 20 μL of modified mNPs were mixed with 20 μL of 10 μg/mL SA-HRP and incubated at room temperature for 10 min. The mNPs were then centrifuged and washed with 10 mM PBS 6 times and redispersed in 0.5% (w/v) of casein in 10 mM PBS. The OD450 of the final solution was 2.80, as measured with a Nanodrop spectrometer (Thermo Scientific).

Test for Model Target TS Sequences

The LF strips of 6.0 cm × 0.5 cm were assembled with a 2.5 cm × 0.5 cm NC membrane (90CNPH-N-SS40, mdi Membrane Technologies, PA, US); 1.5 cm × 0.5 cm absorbent pad (Grade 17 Chr Cellulose Chromatography Papers, GE Healthcare Life Sciences, MA, US); 1.1 cm × 0.5 cm conjugate pad (Grade 6613H, Ahlstrom North America, GA, US); and 1.7 cm × 0.5 cm sample pad (Sample Pad Type GFB-R4, mdi Membrane Technologies, PA, US) on a plastic backing card (mdi Membrane Technologies, PA, US). The NC membrane was fixed at 1.3 cm from an end of the plastic backing card, then at the same end the absorbent pad was fixed. At the other end of the NC membrane, the conjugated pad was attached, followed by the fixation of the sample pad. Between each component there is a 0.2 cm overlap.

On the strips, 0.8 μL of the antibody solution was applied at the center of the NC membrane. The strips were then dried at 37 °C for 40 min and placed on a 3D-printed device fixed with a N52 rare earth magnet beneath the section of the strip where the antibody was applied. To 100 μL of sample solution with model TS sequences at various concentration, magnetic probes were added and incubated for 10 min. Then, 1 μL of CS-FITC was added to the sample solution which was then loaded on the LF strip. After 13 min of sample flow, the strips were washed with 60 μL of DI water every 5 min twice in a cross-flow direction from a conjugate pad at 1.1 cm × 1.1 cm to an absorbent pad at 1.3 cm × 1.3 cm, which were placed on both sides of the LF strip, respectively. Then, 60 μL of the TMB solution was added and incubated for 5 min at room temperature for colorimetric signal generation. The strip was washed with 60 μL of DI water to stop the reaction and remove the colorimetric product caused by the unbound magnetic probes. The results were recorded with images of the strips. The analysis of the color signal was performed with ImageJ (National Institutes of Health, US) and brightness and contrast images were first normalized and then turned to monochrome. The grayscale of the color at the test zone was determined with ImageJ and quantitatively evaluated after the subtraction of the average grayscale of the blank region of the strip as the background.

Test for 16S rRNA from Target Pathogens

Total RNA was extracted from target pathogens and the total concentration was determined with a Nanodrop spectrometer. Then, the stock solution of total RNA was diluted to serial concentrations in 10 mM PBS. The detection of target 16S rRNA in the total RNA from target pathogens was similar to that of model TS. However, due to the secondary structure of 16S rRNA, the hybridization between target 16S rRNA, PS conjugated on magnetic probes, and CS-FITC would be more difficult than that with the short model TS. Therefore, to test 16S rRNA, the CS-FITC would be added along with the probes and the incubation time increased from 10 to 15 min. The rest of the procedure is the same as that of model TS.

To extend the testing methods, we extended our application to detecting pathogenic bacteria, for example, the E. coli contamination of food systems. As an example, lettuce samples were prepared based on the method reported earlier.[47] Typically, 25 g of lettuce obtained from a local market was blended for 1 min in 225 mL of 10 mM PBS. The large particulates in the mixture were removed by a simple filtration and the lettuce solution was next inoculated with serial concentrations of RNA extracts. The detection of 16S rRNA in the lettuce solution was the same as that in PBS buffer.