Magnetic Bacteriophage‐Engineered Janus Micromotors for Selective Bacteria Capture and Detection

Herein, T4‐bacteriophage‐funcionalizated magnetic Janus micromotors are used for the first time for highly selective Escherichia coli (E. coli) recognition and detection. The micromotors propel at speeds of up to 40 µm s−1 in complex biological samples for on‐the‐fly capture of E. coli “strain B”‐thiolated T4 bacteriophage complex. Detection is achieved by a simplified colorimetric readout in connection with gold nanoparticles. The detection limit meets the cut‐off for the fast diagnosis of urinary tract infections. The bacteriophage‐engineered micromotors are tested on bacteria isolated from urine samples and in serum isolated from negative blood cultures from hospital patients with excellent selectivity and reliability. The new strategy described here holds considerable promise for the multiplexed detection of a myriad of bacteria strains using tailored bacteriophages. Technically, this is the first microplate‐reader integrated micromotor approach, crossing another bridge from the research lab to the practical use of micromotors. Specifically, these results represent a qualitative step forward in the use of micromotor technology with sophisticated functionalization for fast bacteria screening in clinical settings.


Introduction
Accurate and prompt detection of bacterial infections is of extreme importance for healthcare systems worldwide. [1]If not treated promptly, patients can develop sepsis, a serious illness with ≈30-40% mortality rates. [2]The gold standard bacterial blood culture method requires days to obtain a result. [3]As such, patients are treated with broad-spectrum antibiotics, leading to antibiotic resistance and inadequate treatments that delay recovery. [4]olymerase chain reaction or ELISA procedures can reduce the analysis time to hours but are still relatively expensive. [5]1a,6] Micromotors are microparticles with sizes ranging from 2 to 20 μm that can move autonomously in solution. [7]Micromotors are promising due to their versatility for modification either on the outer surface using molecularly imprinted technology [8] or functionalization with specific antibodies, lectins, or aptamers for targeted capture. [9]The inherent moving features allow micromotors to operate in ultralow sample volumes with high efficiency, being thus particularly promising for bacteria detection. [10]A pioneering report by Wang's group illustrated the application of concanavalin A-gold-nickelplatinum tubular catalytic micromotors for E. coli isolation. [11]n/Pt Janus micromotors modified with self-assembled monolayers are capable of autonomous loading/release of E. coli bacteria in methanol or p-benzoquinone fuel solutions. [12]Janus fiber rods modified with catalase and mannose have been used for fluorescence detection of E. coli (from 10 2 to 10 5 CFU mL −1 ) via an aggregated-induced emission mechanism. [13]To avoid the use of toxic fuels -which can compromise bacteria viability-ultrasound-driven Au-Ni-Au nanomotors were functionalized with Concanavalin A and antiprotein-A antibody for selective capture of E. coli and Staphylococcus aureus (S. aureus) bacteria, respectively. [14]Pumera's group employed immunoglobulin G modified magnetic micromotors to capture S. aureus bacteria from milk (3.5 × 10 4 CFU g −1 ). [15]Our research group reported the functionalization of magnetic graphene oxide/Fe 2 O 3 Janus micromotors with the antimicrobial peptide Nisin for selective isolation and inactivation of S. aureus in serum samples. [16]Glycomicromotors, prepared by copolymerizing 2-methacrylamidoglucopyranose and 2-(diethylamino)ethyl methacrylate monomers, have been modified with silver nanoparticles for UV-induced electrophoretic movement.The built-in carbohydrate functional groups of the micromotors facilitate its interaction with sugar groups in the membrane of E. coli bacteria. [17]While promising, the previously developed micromotors based strategies focused only on bacteria isolation or lacked selectivity due to the use of non-specific probes such as lectins or carbohydrates.Another interesting approach described by Wang et al. [18] reported on the use of biocompatible high-motility Janus particles that can accumulate passive beads at the surface in the form of clusters and then release them in a controlled manner (under the action of light) at the intended location.Such a new concept can be applied for bacteria isolation and transport but lacks the specific targeting ability due to the absence of a specific receptor.On the other hand, bacteriophages are a type of viruses that can infect bacteria but are harmless to humans.Their structure is relatively simple, with a protein head that encapsulates DNA or RNA genome and a coil.While its main use is the disruption of biofilms, its ability for specific recognition of bacteria has been used for the development of biosensors.An added advantage is the higher stability and low cost of bacteriophages as compared with antibodies. [19]iven the advantages of micromotors and bacteriophages for bacteria detection, herein we describe a biorecognition approach using T4 bacteriophage-modified magnetic Janus micromotors.The micromotors are prepared by a self-assembly approach, using 20 μm polystyrene microparticles as base particles for the modification with graphene oxide, magnetic ferrite nanoparticles, [21] and T4-bacteriophages.The functionalized micromotors can efficiently move in raw samples (urine and serum) for on-the-fly capture of E. coli "strain B"-thiolated T4 bacteriophage complex, generating a sandwich structure.A gold nanoparticle solution (AuNPs) is used for simple colorimetric detection using a microplate reader.In the following sections, we will describe the crucial role of micromotor movement in the detection.The strategy is highly selective, as will be illustrated using S. aureus as interfering bacteria, as well as Klebsiella pneumoniae (K.pneumoniae) and E. coli (a different strain of B) bacteria which were isolated from real urine samples from infected patients.Successful bacteria detection was achieved in urine and serum samples, even in serum isolated from negative blood cultures from patients.The new strategy described here can be extended for the detection of a myriad of bacteria strains using bacteriophages and even in multiplexed assays.To the best of our knowledge, this is the first time that micromotors are combined with bacteriophages for highly selective bacteria detection.Compared with previous micromotor works -either using tubular or Janus designs-the bacteriophage-micromotors reported here present several advantages such as the use of highly selective probes (bacteriophages), which are specific among bacteria serotypes, and the use of magnetic fields for propulsion with enhanced biocompatibility with biological samples.Additionally, the specific size of the Janus micromotors used here (20 μm) is ideal to promote bacteria capture (2 μm long) due to the relatively high micromotor/bacteria ratio sizes, which allow for efficient and improved capture.An added technical advantage is the integration of the strategy in microplate readers, without the requirement for the use of high-performance optical microscopes, holding considerable promise for practical analysis.

Bacteriophage-Micromotor-Based Strategy
Figure 1a illustrates the strategy for E. coli "strain B" detection using bacteriophage-engineered micromotors.In the first step (1), cysteamine modified T4 bacteriophages (for details, please see the Experimental Section) are incubated with the sample or bacteria cultures for 30 min.After washing by centrifugation, on a second step (2) the bacteriophage-functionalized micromotors were added to the previous solution for on-the-fly capture of E. coli "strain B"-thiolated T4 bacteriophage complex, generating a sandwich structure.Magnetic propulsion is achieved by setting the magnetic device at 1600 Hz for 40 min.Additionally, micromotor speed can be controlled using the magnetic set-up described in the experimental section and Figure S1 (Supporting Information).In the third step (3) AuNPs are added for simple colorimetric detection using a common microplate reader.At high bacterial concentrations (300 × 10 6 CFU mL −1 ) the thiol groups present in the bacteriophage induce aggregation of the AuNPs, resulting in a large decrease in the absorbance and color of the solution, allowing modulated detection over a wide range of bacterial concentrations due to the different aggregation of the AuNPs depending on the number of bacteria.Indeed, at low bacteria concentrations (0.3 × 10 6 CFU mL −1 ), the low number of thiol groups (due to less bacteria-thiolate T4 bacteriophage complex captured), prevents AuNPs aggregation, resulting in a lower decrease in the absorbance (see Figure 1B).This aggregation is reflected in the transmission electron microscopy (TEM) images of Figure S2 (Supporting Information), which shows a clear aggregation of the AuNPs in the presence of the modified bacteriophages.The bacteriophage magnetic micromotor strategy combines enhanced micromotor movement with specific target recognition for highly efficient and fast bacteria detection.Figure 1C and Video S1 (Supporting Information) show how the efficient modified micromotor propulsion under the action of magnetic fields, reaching speeds of up to 40 μm s −1 applying a voltage of 4 V, generates a magnetic field of 24 G (Table S1, Supporting Information).Figure 1D illustrates the SEM images of the modified micromotors with the captured E. coli on its surface.The T4 bacteriophage structure consisted of an icosahedral capsid containing the DNA (for additional bacteriophage characterization, please see the UV/VIS spectra and TEM image of Figure S3, Supporting Information).The capsid is connected to a tail tube, by a small structure called a collar, which is surrounded by a contractile sheath.Long tail fibers (LTF) and a special base plate, responsible for the interaction of the bacteriophage with the bacteria membrane, are bonded to the sheath.LTFs can protrude from the phage tail ≈1000 Å and are composed of four different proteins or gene products (gp): gp34, gp35, gp36, and gp37. [20,22]The cell wall of E. coli is formed by a peptidoglycan layer and an outer membrane of lipopolysaccharides and phospholipids.The gp37 (or phage adhesin) interacts with glucosyl-−1,3-glucose disaccharide localized at the core of E. coli "strain B" lipopolysaccharide, resulting in a highly specific and strong bond, responsible for the capture of the bacteria complex with Figure 1.A) Schematic of the bacteriophage-functionalized magnetic Janus micromotors for E. coli bacteria biosensing: 1) Generation of the bacteriacysteamine T4 bacteriophage complex.2) Incubation of the bacteria-T4 bacteriophage complex with the magnetic phage-modified micromotors.3) On-the-fly bacteria capture with the micromotors and addition of the AuNPs.The assay was performed in 96 well plates, following measurements with a microplate reader.B) UV-vis spectra of the AuNPs added to the incubated micromotors, with increasing bacteria concentration.C) Time-lapse images (taken from Video S1, Supporting Information) illustrating the magnetic motion of the micromotors and corresponding tracking lines.D) Scanning electron microscopy (SEM) images of the micromotors after bacteria capture at two magnifications.E) Time-lapse images (taken from Video S2, Supporting Information) showing a micromotor approaching, capturing, and transporting an E. coli bacterium.Scale bars, 10 μm.our micromotors. [23]The recognition event is clearly illustrated in the time-lapse images of Figure 1E and Video S2 (Supporting Information).The modified micromotor approach, interacts and rapidly captures the bacteria, transporting it in a controlled manner without releasing it, allowing for further colorimetric detection in connection with AuNPs.
The images in Figure 1E and Video S2 (Supporting Information) were performed to mimic the micromotor-bacteria recognition event.To this end, we placed the bacteriophage-modified bacteria and the bacteriophage-modified micromotors in a glass slide at the top of the objective of a high-resolution optical microscope.After adequate visualization of a target bacteria and a micromotor, the four-electromagnet-based set-up (for more details, see the Supporting Information) was activated to initiate micromotor motion.Next, with an external magnet, we directed the motion of the micromotor toward the bacteria to achieve the "capture" event.After that, the micromotor propels around the solution to prove that the transport is effective, and the bacte-ria is retained on the micromotor surface (transport step).Please note here that in the detection using the multiwell plate, the micromotors will move randomly in the solution by the action of the electromagnetic field applied with the device, approaching the bacteria present and interacting with the target probe via the bacteriophage-bacteria event.However, it should be noted that this random movement of the micromotors in the plate reader does not turn out to be any inconvenience, given that the use of the multiwell plate represents an instrumental advantage for the future use of micromotors in clinical practice.

Micromotor Synthesis, Characterization, and Magnetic Propulsion
The magnetic motion of the micromotor plays an important role in fast detection, as will be further illustrated.Thus, adequate micromotor synthesis and characterization are essential for further application.Figure 2A shows a schematic of the synthesis (for more details, please see the Experimental Section).Polystyrene nanoparticles (20 μm) were used as base particles for the deposition of a ≈50 nm gold layer by sputtering 1).Next, the microparticles were incubated with thiol modified graphene, promoting attachment to the gold layer via a thiol bond 2).The amount of graphene used was previously optimized to leave a small, exposed gold area for further asymmetric decoration with the magnetic Fe 2 O 3 nanoparticles. [21]A fixed number of the resulting micromotors (≈10 5 micromotors mL −1 ) were mixed overnight with the T4 bacteriophage solution (4 × 10 10 virions mL −1 ), that interact with the ─COOH groups present in the graphene oxide 3).As such, the ─NH 2 groups present on the capside/bacteriophage head link by covalent attachment with the ─COOH groups in the micromotor surface.Thus, the tail of the bacteriophage will have free groups to interact with the bacteria. [24]he asymmetric morphology of the micromotors is reflected in the SEM and EDX images of Figure 2B, which illustrates the C (from graphene) element covering all the micromotor, with an asymmetric Fe part (corresponding to the Fe 2 O 3 nanoparticles).Additionally, to check the successful incorporation/modification of the micromotors with the different materials, SEM images of PS particles, PS/Au particles, and PS/Au/graphene particles were also taken.For EDX mapping, an area of the different particles was selected for mapping.As can be seen in Figure S4 (Supporting Information), in the case of PS particles, only C and O were present.After modification with Au, this metal was observed in the mapping.Please note in the SEM image of the figure the change in morphology, which reveals the Au layer.After modification with graphene, a clear layer is observed, with more abundance of C element.To check the stability of the micromotors, we evaluated both unmodified and modified micromotors.Micromotors stability was checked for morphological changes, adequate functionalization, and detection performance.Unmodified Janus micromotors (without bacteriophages) were stable for 1 month, with changes in the morphology after that period such as the damage/disappearance of the graphene layer, which prevented its functionalization and subsequent detection.The bacteriophage-modified Janus micromotors were stable for 2 days.After this period, the detection signal decreased greatly, indicating the loss of stability in the micromotorbacteriophage union.Please note that it allows for storage of both unmodified micromotors and bacteriophages (by themselves as elements of biorecognition, at −20 °C for 2 months see also Section 4), to functionalize the micromotors before the assays, as needed.
The micromotor can move under the action of magnetic fields (using a tailor-made set-up with four electromagnets controlled by Arduino assembled in our laboratory), as illustrated in the time-lapse images of Figure 2C.The speed can be tailored by controlling the intensity of the magnetic field, reaching remarkable speeds of up to 39 ± 2 μm s −1 at 3.5 V, as can be seen in Figure 2D, which shows the dependence between the applied voltage and the speed of the micromotor (for more information see Table S1, Supporting Information).The biocompatibility of the micromotors was also evaluated due to their application to bacteria biosensing and in biological media.As can be seen in Figure 2E, the micromotor is highly biocompatible (see bar 8), as revealed in the viability of HeLa cells in a common MTT assay.

On-The-Fly Bacteria Capture with the Bacteriophage-Micromotors
The dual role of the bacteriophage modified micromotors for specific bacteria recognition and accelerated capture in microvolumes of the sample was investigated by SEM observation of the captured E. coli bacteria in the micromotor surface (see Figure 3).No apparent bacteria capture is observed performing the experiments using non-modified moving micromotors and modified micromotors in static conditions, revealing specific bacteria recognition ability and the crucial role of magnetic propulsion in the detection (Figure 3A,b,c, respectively).This is further supported by the SEM images obtained after performing the experiments with modified micromotors and using an external stirrer to move the solution (Figure 3A,d), with a very low density of E. coli bacteria captured, as compared with the high-density present in experiments performed with the magnetic moving micromotors (Figure 3A,e,f).This data was further confirmed by UV/VIS measurements, following the detection procedure described in Figure 1A.The data was calculated as normalized absorbances, subtracting the value of the AuNPs solution in contact with the micromotors from the absorbance of the initial AuNPs solution (which was adjusted to 1).Please note here that the modified micromotors, due to scattering, display a background absorbance, [25] which cannot be attributed to the sample (in other words, is not produced by the bacteria capture event).Such background absorbance was subtracted from the signal obtained.Thus, the plot in Figure 3B shows the role of the magnetic micromotors in bacteria capturing and detection.Higher sensitivity (or higher normalized absorbance) is obtained in all concentrations and all cases, using the moving magnetic micromotors.Interestingly, for the lower concentrations tested (3.0 × 10 5 CFU mL −1 ) no signal is obtained using modified static or external stirred micromotors, further revealing the potential of micromotors for targeted bacteria capture in small volumes, even at very low concentrations.This implies that we could detect bacteria early in the infection (due to the high sensitivity) in less time with a smaller sample volume than standardized in routine clinical tests.Additionally, the use of Janus particles (as compared with traditional magnetic nanoparticle-based assays) possesses many advantages as it allows the combination of two functions (propulsion and sensing) with different chemical properties in a single unit toward multifunctional particles.In the case of our micromotor, the asymmetric modification with magnetic Fe 2 O 3 nanoparticles allows for the micromotor movement not only by magnetic rotation but also with a directional thrust, which allows for efficient propulsion in the samples, enhancing the likelihood of contact with the desired target bacteria for enhanced detection in sample microvolumes, in an autonomous way, as it was seen in Figure 3.To sum up, the Janus structure imparts the micromotor with multifunctional properties, combining high specific recognition capacity and efficient "wireless" propulsion in micro volumes of samples for enhanced detection.
In addition, Janus micromotors exhibited a high biocompatibility and have an optimal size for efficient functionalization to capture the bacteria.Please note that the size of E. coli is ≈2 μm long, preventing the use of common tubular micromotors, with an average size of 10 μm, which will minimize the capacity for bacteria capture, as the physical surface available for interaction is narrow.

Analytical Capabilities of the Bacteriophage-Based Micromotors for Bacteria Detection
Prior evaluation of the detection capabilities of magnetic bacteriophage-engineered Janus micromotors for E. coli capture/biosensing in relevant biological media, we optimized the incubation time of the bacteriophage modified micromotors bacteria complex with the AuNPs and number of moving micromotors.As can be seen in Figure S5 (Supporting Information) A, although the highest normalized absorbance (and the highest sensitivity) was obtained after 24 h of incubation of the micromotors with AuNPs, good sensitivities were obtained with only 1.5 h of incubation.Yet, to assess successful functionalization, 24 h incubation was chosen.Please note here that good signals are also obtained (≈20% lower) after only 5 min of incubation with the AuNPs solution.However, as the selective detection of low bacteria concentration is of extreme importance, we selected 1.5 h incubation for the detection.Yet, if fast detection is required, 5 min can be selected, with a total detection time of 45 min (as compared with days using traditional bacteria culture).The optimum signals were also achieved using 1.3 × 10 5 micromotors mL −1 (see Figure S5,B, Supporting Information), which was then selected as optimal.Then, detection capabilities were evaluated.The data from the normalized absorbance for the detection of E. coli fits an exponential model, with a correlation coefficient value of 0.990 (Figure 4Aa,b).The range spans from (0.3 to 300) x 10 6 CFU mL −1 of E. coli bacteria.The limit of detection (1.0 × 10 5 CFU mL −1 ) is higher than that reported using Janus fiber rods micromotors for fluorescence detection of E. coli (from 10 2 to 10 5 CFU mL −1 ), [13] bacteriophage T4-modified magnetic-fluorescent microparticles coupled with flow cytometry (10 4 CFU mL −1 ) [26] or AuNPs and thiolated bacteriophages (10 2 CFU mL −1 ). [27]Yet, our limit of detection meets the level requirements of bacteria established as the cut-off for the diagnosis of Urinary Tract Infections (UTIs, 10 5 CFU mL −1 ) [28] in less than 3 h and in microvolumes samples, holding thus considerable promise for further practical use.
To test the future applicability to complex samples, where concurrent bacteria is common, we tested the strategy for the detection of S. aureus.In this way, it is possible to determine the selectivity of the approach, or the ability of the micromotor to capture and determine the specific bacteria in mixtures with other bacteria without interferences.As can be seen in the SEM images and the plot of Figure 4B,b, no signal is detected using different concentrations by applying the strategy in solutions containing 0.3 × 10 6 , 3 × 10 6 , or 30 × 10 6 CFU mL −1 of S. aureus, as compared with the high normalized absorbance values of E. coli.For 300 × 10 6 CFU mL −1 of S. aureus, there is a light increase in the absorbance, probably due to unspecified absorption because of the relatively high concentration of bacteria.Next, we forti-fied urine and serum samples with increasing concentrations of E. coli, to test the applicability of the strategy in complex samples.For further details, please see the Experimental Section.In urine samples, as can be seen in Figure 4C, recovery percentages ranged from 96% to 100% which indicates efficient bacteria capture as can be also seen in the SEM image of Figure 4C,c, holding thus considerable promise for UTIs detection in routine use without the need for bacteria cultures or other complex method.In serum samples, as can be seen in Figure 4C,b, recovery percentages at 3 × 10 5 CFU mL −1 are ≈80%, with a great decrease (37%) at the high concentration assayed.Non-quantitative recoveries were obtained in the rest of the concentrations tested (data not shown).This data reflects the high complexity of the samples, with a high content of proteins and other interferences, which can induce biofouling and block the active groups from interacting with bacteria.Yet, as reflected in the SEM image of Figure 4C,d, efficient E. coli capture can be achieved at low concentrations of such bacteria.Please note here the good results obtained in the analysis of urine samples (recoveries close to 100% in all cases) at the different concentrations assayed testify to the accuracy of the method for the diagnosis of urinary tract infections.As for the serum samples, good recoveries were obtained at low bacteria concentrations too.
Please note that analytical methods that present low limits of detection for bacterial infections often induce false positives because of the presence of natural bacterial flora in the human body.We consider that our approach can help to avoid this problem because is sensible enough to detect a possible real infection, but not as much to cause false positives.Indeed, these values are compatible with real infection and not with transient bacteremia that are not pathological or with sample contaminations.The excellent selectivity of our method, provided using specific bacteriophages, also contributes to avoiding false positives. [29,30]igure 5. Hospital samples analysis.A) Analysis of E. coli (samples 1,2) and K. pneumoniae (samples 3,4) isolated from patient urine samples B) Analysis of E. coli "strain B'' in fortified serum isolated from aerobic blood cultures (3×10 6 UFC mL −1 ).Error bars represent the standard deviation of three measurements.

Hospital Samples Analysis
To further test the applicability in clinical samples, bacteria isolated from the urine of patients with suspicion of urinary tract infection and serum isolated from blood cultures from patients with suspected infection were analyzed.The results are depicted in Figure 5.The samples from the patients were obtained from Hospital Clínico San Carlos, following all the ethical recommendations and regulations.First, to test the selectivity of the approach, E. coli and K. pneumoniae isolated from real urine samples were analyzed.For details on the sample collection and preparation, please see the Experimental Section.
As can be seen in Figure 5A, two urine samples (samples 1 and 2) from patients with E. coli urinary tract infections were collected.The samples were previously analyzed by the hospital (for additional details, please see the Experimental Section), finding uropathogenic E. coli as the main responsible pathogen causing the infection.In all cases, the concentration of bacteria in the sample is equal to or higher than 10 5 CFU mL −1 .Uropathogenic E. coli compromises a myriad of strains (K-12, CFT073, etc). [31]uch strains are different from our target Strain B, which cannot be found in infected samples since is used for research purposes to guarantee laboratory safety.From the analysis of such samples with the bacteriophage micromotor-based strategy, no response, or a response lower than that noted for the calibration plot performed (denoted as water, reference control) was obtained, indicating that the micromotor does not capture the bacteria due to it belongs to a different strain.These results further indicate the high selectivity of our procedure, since the bacteria present in this sample belong to a different strain.Additionally, another two urine samples infected with K. pneumoniae (samples 3 and 4), were also tested, with any signal recorder, as expected.Overall, these results evidence the excellent selectivity shown by our micromotor approach, even in the case of the same species.Next, to test the feasibility of the strategy in serum, fortified serum isolated from aerobic blood cultures from patients was also tested at 3 × 10 6 CFU mL −1 (Figure 5B).We selected for the analysis blood cultures from patients with a suspected infection but after a routine analysis were confirmed as negative.These negative blood cultures were treated to isolate serum which was fortified with E. coli "strain B" to demonstrate the applicability of our method.As can be seen in Figure 5B, excellent recoveries close to 100% were obtained, testifying to the practical applicability of our approach for bacterial contamination in serum.While selective detection of bacteria is of paramount significance, it must also be considered that absolute values of bacterial concentration are not always informative because the diagnosis of bacterial infection is a very complex process that also includes other multiple factors parameters, such as patient symptomatology, other blood protein biomarkers levels, among others and a final medical diagnosis is highly required.As such, the micromotors can be modified with different bacteriophages for specific bacteria strains related to infection for multiplexed analysis in clinical laboratories, fully integrated into microplate readers.Indeed, it should be mentioned here that the strategy is performed in 96-well ELISA plates and using a commonly available microplate reader, allowing its use in clinical practice, with high expeditiousness.
Pioneering works illustrated the capture of bacteria using catalytic micromotors propelled either by catalytic or ultrasound fields modified with lectins.Such approaches employed lectins as probes, which are less expensive and easier to handle as compared with bacteriophages.Yet, as compared with our approach, the selectivity is not optimal, as unspecific sugar residues in the bacteria membranes are targeted.Additionally, no quantification was performed. [11,14]Glycopolymer-modified Ag/Cl Janus micromotors represent a simplified system combining light propulsion with bacteria capture abilities.As in the previous case, this model is less complex as compared with our micromotor approach.Yet, suffers from selectivity issues, and no quantification is performed. [19]Mannose-modified micromotors combined with tetraphenylethene as fluorescence probes and propelled by catalase have been used for E. coli detection based on aggregated induced emissions.A limit of detection of 45 CFU mL −1 was obtained within 1 min, which is lower than that reported by our method. [13]Yet, peroxide is needed for propulsion, hampering thus application in biological samples due to inherent damage to the biological probes.In another approach, Pumera's group used magnetic micromotors modified with anti-rabbit IgG for S. aureus isolation from milk, which is comparable to that reported in our micromotors. [15]

Conclusion
Herein we have successfully designed and developed for the first time the functionalization of magnetic Janus micromotors with bacteriophages for highly selective bacteria detection under magnetic motion and guidance.The magnetic motion approach avoids the use of toxic peroxide fuel, increasing the biocompatibility with the sample and avoiding further interferences in signal readout.The magnetic features facilitate the manipulation and cleaning steps, allowing to integration of the strategy in ELISA plates and microplate readers for simple readout.To the best of our knowledge, this is the first microplate-reader integrated micromotor approach, crossing another bridge from the research lab to potential routine use in clinical settings.
The selectivity of the strategy was illustrated in clinical samples using bacteria isolated from infected urine samples and fortified serum (with E. coli "strain B") isolated from negative blood cultures, with excellent reliability, testifying to the applicability of the approach in real clinical settings.While reliable bacteria biosensing meets the cut-off value for the urine diagnosis of UTIs (1.0 × 10 5 CFU mL −1 ), it was highly promising for infection diagnosis in complex serum samples too.
Although a myriad of bacteriophages are commercially available, the main limitation is the requirements for highly specific bacteriophage probes, which can be difficult to obtain for highly specific bacterium types.This is a difficult technical issue that requires collaboration among scientists from many fields such as biomedicine, biochemistry, analytical chemistry, and medical doctors.Yet, specific infection in hospital with highly specific bacteria needs previous identification by a medical doctor.Next, technologies such as chemical synthesis, and phage engineering, among others, should be tested to get a bacteriophage with the desired selectivity for a specific bacterium.Also, as biological probes, the bacteriophages are not as stable as lectin-based approaches, but this is also a common problem when using antibodies and other biological probes.As we have already indicated on other occasions, micromotor technology continues to await the advances of others to continue its growth and establishment in areas of extraordinary importance such as biosensing, biotechnology, and precision biomedicine.
However, critically, in this frame, the new creative approach described here holds considerable promise for the highly selective targeted detection of a myriad of bacteria strains using tailored bacteriophages and even, in multiplexed assays.

Experimental Section
Bacteriophage Culture and Filtration: Escherichia coli (E.coli) "strain B" cells were grown under agitation in 15 mL of LB medium at 37°for 5 h using a 50 mL sterile conical tube.Next, four aliquots were added to four different tubes, and a bacteriophage culture was added to each one.The tubes were incubated under agitation at 37°for 2 h.Then, LB medium was added to fill up the tubes which were incubated under agitation at 37°overnight.The next day, the tubes were filtered to remove the alive and lysed E. coli.First, the solution was filtered through a 0.45 μm cellulose nitrate filter membrane and, after that, the liquid, which contains the bacteriophages, was filtered again using 0.20 μm sterile syringe filters.Bacteriophage suspension was finally filtered using tubes provided with a 100.000Da membrane by centrifugation (500 RCF x 10 min).This process was repeated until all the liquid was filtered and then, filter-retained bacteriophages were washed three times with Milli-Q water using the same centrifugation method.Finally, 3 mL of water was added to the filters and the clean bacteriophages were resuspended using a micropipette.The final concentration of the bacteriophages of this 3 mL of water was calculated using a microplate reader, making a 200-400 nm absorbance spectrum.The result was obtained by applying the Equation (1). [32]rions mL = (A 269 − A 320 ) × 6 × 10 16 number of bases of the virion (1) The bacteriophage solution was stable at −20 °C for 2 months without the need for another incubation process.
Thiolation of Bacteriophages: 1 mL aliquot of the bacteriophages was shaken overnight with 30 mg of EDC and 20 mg of cysteamine.The next day, the thiol-modified bacteriophages solution (bacteriophages-SH) was filtered by centrifugation two times (4300 RCF x 5 min) using 10K Amicon ultrafilters.After that, the filters were cleaned with Milli-Q water by centrifugation.Finally, the bacteriophages-SH were resuspended in 1 mL of Milli-Q water and stored at 4 °C until use.These solutions remain stable for 2 days.
Synthesis of AuNPs: For the synthesis of the AuNPs (4 nm, see TEM images of Figure S2, Supporting Information), gold (III) chloride trihydrate 5 mm, sodium citrate 5 mm, and sodium borohydride 5 mm.First, a 20 mL beaker and a magnet were cleaned using a 3 m chlorohydric acid solution for 5 min and washed with Milli-Q water.After that, 18 mL of Milli-Q water was added to the beaker and stirred using a stirrer plate and the magnet which was previously cleaned.Without stopping the agitation, 1 mL of the gold (III) chloride trihydrate solution was added followed by 2 mL of the sodium citrate solution.The resultant solution was shaken for 2 min.As the last step, the sodium borohydride solution was added by 200 μL aliquots to a final volume of 1200 μL.In the end, the solution changed its color from colorless to brilliant red.
Micromotor Synthesis and Characterization: First, 20 μm polystyrene particles were dropped in two microscope slides (76 mm x 52 mm) and covered with a ≈50 nm gold layer.Once metalized, the polystyrene particles were scraped and resuspended in 8 mL of Milli-Q water.To prepare thiol-modified graphene (GO-SH) solution, 10 mL of 1 mg mL −1 graphene-oxide (GO) solution, 10 mg of cysteamine, and 20 mg of EDC were mixed overnight.The next day, the GO-SH solution was washed three times by centrifugation (6700 RCF x 5 min) and resuspended in Milli-Q water (10 mL).Additionally, 10 mL of 1 mg mL −1 Fe 2 O 3 solution was obtained by dissolution of Fe 2 O 3 nanopowder in Milli-Q water.To synthesize the Janus micromotor, 950 μL of the polystyrene solution was added in a microcentrifuge tube and mixed with 50 μL of GO-SH solution (dispersed previously in an ultrasonic bath for 5 min) under external agitation for 2 h.After that time, 50 μL of the Fe 2 O 3 solution (dispersed in an ultrasonic bath for 5 min) was added to the tube and mixed for another 2 h.The resultant solution was washed with Milli-Q water using a 5 μm polycarbonate membrane.The clean micromotors were resuspended in 1 mL of Milli-Q water and the concentration of micromotors was adjusted to obtain the concentration final of 10 5 micromotors mL −1 .To achieve this, three drops of 1 μL each were visualized with an optical microscope, and the micromotors were counted.With the average concentration, the final concentration of the milliliter was obtained and adjusted to the desired value.The next step was to attach the non-modified bacteriophages to the micromotors.To achieve this objective, 1 mL of the micromotors was put in contact with 1 mL of non-modified bacteriophages (2 × 10 11 virions mL −1 ) obtained and mixed overnight.Finally, these micromotors were washed by centrifugation three times (6700 RCF x 5 min) and resuspended with Milli-Q water in 1 mL.
E. coli Culture: E. coli "strain B" cell culture was grown under agitation in 5 mL of LB medium at 37°overnight in a 50 mL conical tube.The next day, 1 mL was centrifugated (1100 RCF x 5 min) and the supernatant was discarded.The bacteria pellet was resuspended in Milli-Q water and washed two more times following the same method.Finally, the bacteria pellet was resuspended in 1 mL of Milli-Q water.The estimation of the concentration of colony-forming units (CFU mL −1 ) was done by the measure of OD 600 using a microplate reader, applying Equation (2). [33]U∕mL = ( OD 600 4 × 10 8 ) + ( 2.18 × 10 7 ) (2) Protocol for the Analysis Using the Bacteriophage-Modified Micromotors: Four different concentrations (3 × 10 8 , 3 × 10 7 , 3 × 10 6 , and 3 × 10 5 CFU mL −1 ) of bacteria were obtained making dilutions of the concentrated one.250 μL of each solution were taken and put in contact under agitation with 50 μL of bacteriophage-SH for 30 min.The next step was to clean three times the bacteria and bacteriophages-SH with Milli-Q water by centrifugation (1100 RCF x 5 min) and resuspend the final bacteria pellet in 300 μL.The clean solutions of bacteriophage-SH and bacteria complex were situated on a multiwell plate, which was placed over a magnetic plate stirrer, set at 4000 Hz.Another magnet was located over the multiwell plate to avoid the precipitation of the micromotors to the bottom of the well.After 40 min of propulsion, an NdFeB magnet was placed under the multiwell plate to hold down the micromotors with the bacteria attached to them.The supernatant was discarded and 300 μL of Milli-Q water was added.This process was repeated two times to clean the micromotors and discard the unattached bacteria.Finally, the last 300 μL of water was discarded using the NdFeB magnet to hold down the micromotors and 300 μL of the AuNPs was added and incubated with the bacteria-attached-micromotors for 90 min at room temperature.After that time, the NdFeB magnet was located again under the wells and the nanoparticle solutions were situated in clean wells to prevent the micromotors from interfering with the subsequent measurement.To determine the aggregation of the nanoparticle solutions and, therefore, the concentration of bacteria, the absorbance was measured using a microplate reader from 400 to 800 nm (step: 1 nm).Measurements of AuNPs that had been in contact with modified micromotors without bacteria and with AuNPs were also done.The data treatment of the absorbance spectrums obtained followed the next process: first of all, the spectrum's baselines of the different concentrations were adjusted to "0" and the peak of the nanoparticle's solution spectrum was considered as "1".The other spectrum peaks, which correspond to the different concentrations of bacteria present in the sample, were adjusted in correlation to the relatively high of the nanoparticles solution.To achieve a correct calibration curve, the absorbance of a solution containing modified micromotors is measured, which due to light scattering produces a background signal, which was subtracted from signals obtained from the different concentrations of bacteria in contact with AuNPs.Finally, the relative height of the peak versus the bacterial concentration present in the sample was represented.With the calibration curve obtained was possible to quantify the real samples.
Serum and Urine Analysis: To obtain the fortified urine, the bacteria culture was done as described above: E. coli cell cultures were grown under external agitation in 5 mL of LB medium at 37°overnight.The next day, 1 mL of the culture was centrifugated (1100 RCF x 5 min).The supernatant was discarded, and the resultant pellet was resuspended in 1 mL of urine, repeating the process two times.The rest of the determination process was the same as the one followed to obtain the calibration curve and the values obtained from the different concentrations of bacteria were compared with the values obtained previously in Milli-Q water.The process was the same with the human serum, but in this case, the commercial serum was previously diluted 1:2 in Milli-Q water.
Interferences Study: The interference study was carried out with Staphylococcus aureus (S. aureus).S. aureus cells were grown overnight under external agitation at 37°in 5 mL of LB medium.The culture was cleaned the next day using the same centrifugation method as used with E. coli calibration and resuspended in 1 mL of Milli-Q water.The estimation of CFU was done by the measure of OD 600 using a microplate reader and adjusting the absorbance to 1, which was considered to correspond to 1.5 × 10 8 CFU mL −1 . [34]ospital Sample Collection and Preparation: Urine samples were obtained from patients with suspicions of urinary tract infection.Samples were collected with a sterile loop and subsequently deposited in an agar plate.After standard culturing, samples were measured using the OD 600 with a microplate reader.Samples were considered positive by the hospital laboratory when the UFC mL −1 was equal to or higher than 10 5 , assuring that the sample was not contaminated by other concurrent bacteria present in the urinary tract or the urethra.In all the samples analyzed in this work, bacteria concentration was >10 5 UFC mL −1 .Once in the lab, bacteria colonies were isolated and cultured overnight as described previously, to get a concentration of 10 6 UFC mL −1 in all samples.
Aerobic blood samples were cultivated in an automatic incubator following measurement of gas production during growing.In this case, the analysis was qualitative, classifying the samples as positive or negative.Blood samples were collected from patients with suspicion of infection by venous puncture.The blood samples were discarded by the incubator system as negative for serum isolation, blood samples were centrifugated at 1600 g for 10 min resulting in the formation of two phases, the bottom one will be the blood cells, and the top one will be serum, which was carefully extracted with a Pasteur pipette.
Magnetic Set-Up for Micromotor Propulsion: The magnetic movement studies were carried out using a 3D-printed device to support an electromagnet (which consists of a copper solenoid).This electromagnet was connected to an adjustable power supply.The voltages were modified between 1.5 and 4 V, allowing for the modulation of the speed of the magnetic micromotors.The magnitude of this magnetic field was obtained empirically, measuring it on the electromagnet surface using a tailor-made device.After that, the magnetic field on the position of the micromotor was calculated.The set-up was assembled according to Figure S1 (Supporting Information), A. Real pictures of the set-up can be seen in Figure S1,B (Supporting Information).
The magnetic field was calculated applying Equation (3), where B x is the magnetic field at a position x from the center of the solenoid (considering a 90 °C angle) and Bo the magnetic field at the center of the solenoid, calculated by Equation ( 4). (5) Statistical Analysis: All the data presented was presented as the mean ± SD of n = 3 analysis.Origin lab software was used for statistical analysis and data display.

Figure 3 .
Figure 3. Influence on the enhanced micromotor fluid mixing on E. coli bacteria capture and detection.A) SEM images of (a) modified micromotors; (b) static modified micromotors in contact with E. coli; (c) nonmodified moving micromotors in contact with E. coli, (d) modified micromotors under external stirrer agitation in the presence of E. coli and (e, f) modified micromotors under magnetic motion in contact with E. coli.B) Corresponding plot showing the absorbance (as normalized absorbance) of the system in the presence of different concentrations of E. coli with the moving micromotors, static micromotors, and external magnetic agitation conditions.Scale bars, 20 μm.Conditions: incubation time, 40 min, 10 5 micromotors mL −1 .Error bars correspond to the standard deviation of three measurements.

Figure 4 .
Figure 4. Detection and analysis performance of the bacteriophage-modified magnetic Janus micromotor bioassays for E. coli detection.A) Absorbance values (a) and corresponding E. coli concentration-dependence plots (in CFU units) (b).B) Selectivity of the assay: SEM images showing the micromotors after contact with E. coli and S. aureus bacteria (a) and corresponding absorbance plots (b).C) Analysis of spiked samples: absorbance values in the presence of different concentrations of E. coli (a, b) and SEM images showing the micromotors after bacteria capture in spiked urine (c) and serum (d).Scale bars, 2 μm.Error bars correspond to the standard deviation of three measurements.