Magnetic plasmonic particles for SERS-based bacteria sensing : A review

This review describes recent advances in the use of magnetic-plasmonic particles (MPPs) for bacteria detection by SurfaceEnhanced Raman Scattering (SERS). Pathogenic bacteria pollution has always been a major threat to human health and safety. SERS spectroscopy has emerged as a powerful and promising technique for sensitive and selective detection of pathogen bacteria. MPPs are considered as a versatile SERS platform for their excellent plasmonic properties and good magnetic responsiveness. Improved preparation method and typical characterization technique of MPPs are introduced, focusing on the thin and continuous metallic shell covering process. Consequently, the SERS-based sensing methods for bacteria identification were discussed, including the label-free and label-based methods. Finally, an overview of the current state of the field and our perspective on future development directions are given. © 2019 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/1.5050858


I. INTRODUCTION
Diseases caused by pathogenic bacteria remain a major threat to safety and health of human beings. 1 Pathogens present in food, water, or even in air often contain etiologic agents of many serious and even fatal diseases.Furthermore, some infectious disease bacteria can broaden the range of pathogen pollution and infect others.Rapid and accurate detection of pathogenic bacteria is therefore considered an effective way to save lives and reduce healthcare costs. 2,3The current gold standard for pathogens testing in hospitals is based on strains culturing and growing, often known as colony counting method.][6] An alternative approach is to detect the specific gene of the pathogen instead of detecting the pathogen itself.Real time polymerase chain reaction (rtPCR) is recently preferred to identify the pathogens in DNAs sequences during their amplification, only taking 1∼2 hours.][9][10] In addition to the laborious pretreatments, rtPCR method suffers from occasional fluorescence quenching, single-channel detection, and sophisticated equipment, as it acquires the fluorescence signals of the labels which are modified in the end of the DNA sequences.Therefore, there is great need to develop novel techniques which can detect the pathogens directly while reducing the assay time significantly.
Surface-enhanced Raman scattering (SERS) is a powerful and promising spectroscopy technique that can not only AIP Advances 9, 010701 (2019); doi: 10.1063/1.5050858 9, 010701-1 © Author(s) 2019 2][23][24] For bacteria, the SERS-based sensing approaches are classified into two categories, namely the label-free and label-based methods. 25,26The label-free approach detects bacterial pathogens without any label because each bacterial pathogen has an intrinsic SERS spectrum.8][29][30][31] Simultaneous detection and identification of various pathogens is realized by analyzing the SERS spectra. 32By contrast, the label-based approach employs a label, usually called SERS-tag, 25 to mark the pathogens.In this scenario antibodies and aptamers are often utilized to form a specific binding to the bacteria and construct a sandwich structure.The SERS signal of the SERS label reports the appearance of the bacteria through the specific bio-recognition.The schematic illustration of label-free and label-based SERS method for bacteria detection is shown in Fig. 1.
Both label-free and label-based sensing approaches rely on the SERS substrate's dramatic signal enhancement ability (10 5 to 10 12 ), which originates dominantly from the electromagnetic field concentration or coupling at the nanotips or nanogaps of the noble metal nanostructures.These areas with greatly enhanced electromagnetic field are called 'hotspots' for SERS and are used to amplify the usually weak Raman scattering signals.The fabrication of high-performance SERS substrate is a key factor for the bacteria detection.By now, SERS substrates or platforms were constructed generally by either solution-based nanoparticles (NPs) synthesis or solid-phase nanostructure (NS) fabrication. 33The solid phase NS substrates, especially the ones with complicated morphologies, have received much attention due to the hotspots engineering at the tips and gaps.6][57] However, sophisticated equipment, tedious process, high cost, and poor biocompatibility of the solid phase substrate hamper its application in bioassay.The solution-synthesis-based NPs, such as Au/Ag NPs, 58,59 core-shell NPs, 11,60,61 self-gapped NPs, 62 and DNA connected dumbbells NPs 63,64 demonstrate remarkable signal enhancement ability, excellent biological compatibility, simple and cost-effective synthesis process.However, the NPs suspension have several drawbacks including the poor stability and difficulty in hotspots control.
As a novel SERS substrate, emerging magnetic-plasmonic particles (MPPs) have attracted increasing attention in recent years due to their excellent controllability and stability, though synthesized in solution.The magnetic core can be conveniently manipulated by the external magnet, which facilitates the process to separate the targets, replacing the usually used centrifugation process.The noble metal shell provides the hotspots in the SERS measurements.With advantages of good sensitivity, simple fabrication procedure, low cost and excellent controllability, MPPs demonstrate great potential and promise in SERS-based sensing, especially for rapid bacterial cell detection.This review is mainly based on the work in our lab conducted in the past five years.A comprehensive literatures are included to provide an overview of current developments and trends in pathogen bacteria sensing with the aid of the MPPs as the SERS substrate.Firstly, we will give an overview of MPPs preparation methods and the typical characterization means.Secondly, we will discuss the use of MPPs in bacteria detection with the label-free and label-based SERS sensing methods.For the label-based SERS sensing, typical immuno-assay and aptamer recognition sensing methods will be discussed separately.Finally, we will outline challenges and future perspectives in this evolving field, such as the sensing reproducibility, whole-organism fingerprinting database establishment, and parameters optimization.

II. MPPs PREPARATION AND CHARACTERIZATION
MPPs usually have a core-shell structure, with the magnetic core inside and the noble metal shell outside.The functionalized MPPs are generally prepared via three steps: the magnetic core synthesis, noble metal shell coating, and bio/chemical modification (Fig. 2a).6][67] Herein, M represents the divalent metal ions, such as Fe 2+ , Mn 2+ , Zn 2+ , Cu 2+ , Ni 2+ , Mg 2+ , Co 2+ .Then, the magnetic core is coated with a thin and continuous noble metal shell, such as Au and Ag, for outstanding SERS performance.To catch and enrich the target, the prepared MPPs is functionalized with chemical or biological molecules.

A. MPPs fabrication background
A myriad of synthetic methods have been proposed to prepare high-performance MPPs with good monodispersity, strong magnetic responsiveness, excellent SERS performance, and good biocompatibility.Zhong's group employed thermally activated processing protocol to prepare MPPs. 68,69The particles obtained have a mean diameter of 6.3nm, which is too small to provide enough magnetic responsivity.These small AuMPs fabricated by Zhong's group are not sufficient for target separation, especially for the big bacterial cells.To fabricate bigger MPPs (diameter>100nm), the AuNPs were grafted directly to the magnetic core either by the chemical bonding 70 or the electrostatic interaction 71 to prepare Fe 3 O 4 -Au NPs composites.However, AuNPs cannot cover the magnetic core, which will evidently affect the composites' SERS performance.To obtain a continuous shell, silicon was employed as an interlayer for the metal shell formation.Ji's group coated the Fe 3 O 4 core with a silica shell to facilitate deposition of gold seeds and reduction of Kgold solution with formaldehyde. 72Han's group coated Ag shells on a Fe 3 O 4 @SiO 2 composite using a "seed-mediated growth" method. 73Hu's group deposited AgNPs on surfaces of Fe 3 O 4 @SiO 2 composite by the Ag-mirror reaction. 74However, the nonmagnetic silicon interlayer seriously affected the magnetic capability of the MPPs.Li's group developed a facile onepot hydrothermal approach to synthesize Fe 3 O 4 @Au NPs. 75he controllability of the process and the uniformity of the synthesized particles however need further improvements.Hydroxylamine seeding method can be utilized to synthesize AuMPs with uniform size and shape because of its separation of nucleation and growth stages. 76Hydroxylamine or hydroxylamine hydrochloride can reduce chloroauric acid to elemental gold (Au 0 ) and gold particle surfaces can accelerate the occurrence of this reaction {Winkler, 2011 #6765}.Reduced Au 0 can be adsorbed on the surfaces of existing Au seeds, thus generating larger NPs rather than new nucleation.Lyon's group modified the hydroxylamine seeding procedure of Natan to synthesize AuMNPs for the first time and named the modified approach as iterative hydroxylamine seeding. 77ased on Lyon's method, Zhou's group 78  Although the iterative hydroxylamine seeding growth method has been widely used, its cumbersome and time-consuming route remains to be optimized.

B. Fabrication
To coat a thin and continuous metal shell on the magnetic core, our group have developed a sonochemically assisted hydroxylamine seeding growth method to synthesis MPPs (Fe 3 O 4 @Au).Firstly, magnetic Fe 3 O 4 particles are synthesized through a modified solvothermal reaction. 67An example transmission electron microscope (TEM) image of the synthesized Fe 3 O 4 particles is shown in Fig. 2b.Secondly, the metal shell of the magnetic core is coated via three steps: a) hydrophilic Polyethyleneimine (PEI) is self-assembled on the surface of Fe 3 O 4 particles as the linkers to form a Fe 3 O 4 @PEI microsphere under the sonication conditions (Fig. 2c).The thickness of PEI layer is controlled by adjusting the sonication time.b) Au seeds (small Au nanoparticles with the diameter of 3∼5nm) are grafted on Fe 3 O 4 @PEI to form monodispersed Fe 3 O 4 @PEI-AuNPs microspheres under the sonication conditions (Fig. 2d).These Au seeds act as the nucleation sites for the subsequent seed-mediated growth of the Au shell.c) HAuCl 4 is reduced by hydroxylamine hydrochloride and deposited on the Au seeds, affording a continuous and homogeneous Au shell around the Fe 3 O 4 core (Fig. 2e).Finally, a PEI layer is modified around the MPPs to justify the surface property of the particles (Fig. 2f).The surface of MPPs can be modified by using other chemical or biological molecules, depending on the nature of the target to detect.

C. Typical characterization methods
The synthetic steps and all intermediates MPPs need to be characterized by a variety of techniques.Apart from standard scanning electron microscopy (SEM) and Transmission Electron Microscopy (TEM) techniques, X-ray diffraction (XRD), UV-visible absorption spectra, superconducting quantum interference device magnetometer (SQUID) characterization, and zeta potential measurements were also used.
The crystal structure and phase purity of the as-prepared products are usually characterized by XRD, as shown in Fig. 3a.The characteristic peaks of Fe 3 O 4 are marked with " * ," and those of Au are marked with "#." Curve A represents a typical XRD pattern of the Fe 3 O 4 particles. 80After the absorption of the Au seeds, a new XRD peak (Curve B of Fig. 3a) can be observed at the diffraction peaks (2θ) of 38.2 • , corresponding to the (111) crystalline plane of Au. 81 After a continuous Au shell forms outside the magnetic core, the diffraction peaks of Fe 3 O 4 are no longer observed in Curve C of the Fe 3 O 4 @Au microspheres, which indicates the "complete coverage of the Fe 3 O 4 core with Au shell." 78e size dispersity and the surface materials of the products are usually characterized by UV-visible absorption spectra, as shown in Fig. 3b.The broad peak band of Curve A illustrates the polydispersity of bare Fe 3 O 4 microspheres, which can be attributed to the ferromagnetic behavior of the magnetic cores. 82When the magnetic core is covered with Au seeds, the peak of the Fe 3 O 4 shows a "red shift," indicating the growth of the particles.However, no additional peak was observed as the adhered Au seeds are too small to affect the plasmon behavior (Curve B).When the Fe 3 O 4 @Au microspheres are coated with a continuous Au shell, an obvious absorbance peak can be observed at 568 nm (Curve C), as a consequence of the surface plasmon coupling between the neighboring Au shells. 83e magnetic properties of products are usually assessed by SQUID, as shown in Fig. 3c.All of the curves nearly intersected with the origin, indicating that the remanence of the particles disappeared rapidly when the external magnetic confinement is removed.Thus, the synthesized particles are in a superparamagnetic state at the room temperature. 80The saturation magnetization values tends to decrease slightly with the non-magnetic materials coating process.The magnetic separation of the MPPs is completed within 10s, even after the functionalization, as shown in the inset of Fig. 3c.The magnetic responsiveness provided by the MPPs are strong enough for target separation.
The surface charge properties of the products are characterized by the zeta potential measurements, as shown in Fig. 3d.After each step of PEI modification, the zeta potential of particles becomes strongly positive due to the cationic nature of the PEI polymer.

D. SERS application
To quantify the enhancement ability of the SERS substrate, the enhancement factor (EF) calculation and the detection limit determination are usually required.
EF is defined as a ratio of photons scattered by SERS substrate and normal substrate, and calculated according to the following formula. 84

EF
Where I SERS and I NR are the Raman signal intensities measured on SERS-active substrate and non-SERS-active substrate (normal Raman), respectively; N SERS and N NR are the numbers of probe molecules contributing to the corresponding Raman signals I SERS and I NR .As an example, the EF of proposed MPPs substrate is calculated to be much higher than 10 7 .
To determine the detection limit, controlled experiments are conducted using the MPPs as the SERS substrate.Herein, p-aminothiophenol (PATP) is used as a probe with concentrations ranging from 10 −5 to 10 −10 M. PATP detection limit of the MPPs substrate is 10 −10 M judging from the vibration band at 1078 cm −1 , as shown in Fig. 4. The error bars in Fig. 4b indicate standard deviations from five measurements for each sample.

III. PLASMONIC CHARACTERISTICS
The Raman signal can be enhanced by 10 7 times or even more (enhancement factor) using the plasmonic particles as SERS substrate.It has been demonstrated that the dramatical enhancement was originated from both the physical mechanism and chemical mechanism.Since the chemical mechanism normally provides about 10∼100 folds enhancement, the physical mechanism plays a predominant role in Raman signal enhancement, which is proportional to the fourth power of the EM field intensity.(Moskovits 1985) The area where the electromagnetic field concentrated or coupled are considered as the main factor that contributes to physical enhancement, which are usually called hotspots.The location and the strength of the hotspots can be investigated by solving the Maxwell equation.There are some sophisticated strategies as well as commercial available softwares for the numerical calculation, such as the FDTD, DDA, and etc.Recently, the FDTD method was the most popular one.
To investigate the plasmonic characteristics of the magnetic particles, FDTD method was employed for the visualization of the electromagnetic field on the surface of the particles.It is well known that single plasmatic particles provide limited EM field enhancement, thus can not form the hotspots.The hotspot usually located at the gap between neighborhood spherical NPs due to the electromagnetic field coupling.Three class of particles were studied, including the AgNPs, MPPs and magnetic particles.To simplify the calculation model, two NPs system were employed herein.To make the hotspot locate at the center of the two NPs, not around the junction, the NPs was fixed to be 1nm apart.
The EM field coupled at the gap between the particles as shown in Fig. 5(a∼c).The EM field enhancement of three particles at the wavelength ranging from 300nm∼900nm was shown in Fig. 5d.The Ag NPs are typical plasmatic particles, which can provide sufficient EM field enhancement at the gap.The MPPs can provide similar EM field enhancement.The magnetic core gave limited effection to the plasmatic characteristic of the particle.The surface plasma was mainly provided by the Ag shell.However, the peak of the enhancement curve was blue shift due to the addition of the magnetic core.The bare magnetic particles provide little EM field enhancement because no surface plasma was excited by the laser.To sum up, the bare magnetic particles can not provide surface plasma, while the addition of magnetic core will not affect of the plasmatic characteristics of the noble metallic particles.

A. Sensing principle
As mentioned, label-free method detects the intrinsic SERS fingerprint of bacteria without using a label, making the process usually fast and relatively simple.][87] Label-free detection method can not only distinguish species of bacteria, 88,89 but can also identify whether the bacteria is live or dead. 90,91Label-free bacteria sensing presents the new frontier of cell-based assays. 85Two main challenges still hinder the wide applications of the label-free method: the sensitivity improvement and the bacteria enrichment.To date, the spectra intensity of the cell wall components is very weak.In addition, the components cannot be positioned right at the hotspots of the SERS substrate, which will seriously affect the sensitivity of the sensing method.Thus, the performance of the SERS substrate is a key factor for label-free bacteria detection method.Moreover, enriching the bacteria and avoiding the interference from the impurities is essential for the real sample testing.Mosier-Boss's group, 92,93 and Evelin Witkowska's group 94 have enriched the bacteria cell on/around the SERS substrate by a filtering process.However such strategy can introduce some interference spectra due to the presence of big molecules in the sample.Yu's group 95  Given these challenges, we have brought out a captureenrichment-enhancement (CEE) method for the rapid and sensitive bacteria detection.A kind of novel MPPs (Fe 3 O 4 @Au microspheres) is synthesized and used as SERS substrate.The MPPs are positively charged by the presence of an aminofunctionalised polymer.Then, MPPs are dropped into the bacteria solution and incubated for 5 min.During this process, the negatively charged bacteria are rapidly captured and separated by the positively charged MPPs under an external magnetic confinement, resulting in the enrichment of the bacteria.The separated composites are consequently dropped on a clean Si substrate and followed by the addition of concentrated Au@Ag NPs to further improve enhancement.The Au@Ag NPs can cover the blank surface of bacteria, and produce more hotspots.The schematic of CEE procedure for the rapid SERS detection of bacterial pathogens is shown in Fig. 6.

B. Bacteria capturing and enrichment
To capture and enrich the bacteria, the amino-groups of the PEI are firstly protonated to afford a positive charge on MPPs surface.This causes a strong electrostatic interaction between the MPPS surface and negatively charged bacterial walls, thus capturing the bacteria in the solution.To evaluate the bacterial capture ability of the MPPs, Escherichia coli.(E.coli.) is used as an example and its optical density(OD) at the wavelength of 600nm (OD 600 ) is measured.The concentration of bacteria is adjusted to a OD 600 of 0.5 at first as shown in Photo A in Fig. 7a.The MPPs with and without PEI-functionalization are added into the E. coli solution and incubated for 5 min, respectively.After the magnetic aggregation, the supernatant solution added with bare MPPs (i.e., Photo B in Fig. 7a) appears nearly the same as that of original E. coli solution (i.e., photo A in Fig. 7a), while the supernatant solution added with PEI modified MPPs becomes nearly transparent (i.e., photo C in Fig. 7a).The OD 600 testing confirms the contribution of PEI layer in bacteria capturing.The capture efficiency (CE) of the PEIfunctionalized MPPs is calculated to be 66.2% following the formula CE (%) = 100(αβ)/α, where α and β represent the OD 600 values before and after magnetic separation. 96OW?? (Fig. 7b).Fig. 7c shows the TEM image of MPPsbacteria complex.The MPPs are conjugated tightly outside E. coli., facilitating Raman signal enhancement as illustrated in Fig. 7d.The major vibrational modes of E. coli can be observed at the concentration of 10 6 cells/mL, as it is captured by the MPPs microspheres, while no characteristic vibrational bands are observed even at the bacterial concentration of 10 8 cells/mL mixed with Fe 3 O 4 @Au and Fe 3 O 4 @PEI microspheres.Capturing the bacterial cells is based on the electrostatic interaction between the positively charged MPPs and the negatively charged bacteria walls.Thus, the amine groups modified on the MPPs are potentially nonselective ligands to various types of bacteria because the cell walls of bacteria are generally negatively charged. 97

C. Sensitivity improvement
To determine the sensitivity of prepared SERS substrate, SERS spectra of E. coli are obtained at different concentrations ranging from 1 × 10 7 cells/mL to 1 × 10 2 cells/mL.The detection limit of MPPs is about 1 × 10 5 cells/mL, insufficient for bacteria sensing.A scattered distribution of AuNPs around the bacteria can be observed in Fig. in Fig. 8d.The detection limit is about 10 3 cells/mL for both Gram-positive bacterium E. coli and Gram-positive bacterium S. aureus, which is comparable to that of the PCR method.The whole process, from the bacteria capture to SERS signal measurements, can be completed within 10 minutes.
The selectivity of this label-free sensing strategy is based on the instinct Raman fingerprints of the bacterial cell.To easily distinguish the spectra of the bacterial, principle component analysis (PCA) method was usually applied. 95PCA method can reduce the dimensionality of multivariate spectral data and group the similar spectral data for classification.Generally speaking, the more samples PCA method include, the more accurate should the distinguish process be.

V. LABEL-BASED BACTERIA DETECTION
Label-based bacteria detection is an extrinsic mode method, which reports the target indirectly by collecting and analyzing signals of the so-called SERS-tag.][100] The typical sandwich structure is usually constructed with the aid of specific ligands such as antibodies and aptamers.Thus, the selectivity of the assay is based on the specific recognition of the ligands.The sandwich structure contains a MPPs as the SERS substrate, a captured bacteria as the target and a SERS-tag as the reporter.Hotspots are created at the junctions between MPPs and SERS-tag at the appearance of target bacteria.

A. Immuno-sensing
The SERS-based immuno-assay for the bacteria is based on the construction of sandwich complex by the specific antigen-antibody conjugations, as illustrated in Fig. 9. Herein, Au coated MFe 2 O 4 particles are utilized as the MPPs, the Staphylococcus aureus (S. aureus) are employed as the target, and the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) marked nanorod is used as the SERS-tag.Preparation of the proposed MPPs is similar to that introduced above, as shown in Fig. 2a.The prepared MPPs are modified with selected antibody and incubated in the S. aureus solution for the specific bacteria capture.The particles containing captured S. Aureus.are then separated by an external magnet, a more effective and simpler method compared to traditionally used centrifugation.The uncaptured bacteria that remains in the supernatant are consequently removed.The second-antibody modified SERS-tag is dropped into the suspended solution to construct the sandwich structure.The bacteria have respective conjugation sites for the antibody and second-antibody, as shown in Fig. 9b.Finally, the sandwich complex is separated and placed on a supporting silicon slice for SERS measurements.
By using an antibody as both recognizer and linker, selective detection of S. aureus is achieved with a detection limit down to 10 cells/mL.To quantify the dose-response of the proposed method, a calibration curve between the SERS intensity at the vibration band at 1331 cm −1 and the logarithm of S. aureus bacteria concentrations (10 1 -10 5 cells/mL) has been plot and is shown in Fig. 10b.A good linear relationship was obtained with a correlation coefficient of 0.9789.The error bars represent the standard deviations from five measurements for each sample.

B. Aptamer recognition sensing
Aptamers are single-stranded DNA or RNA molecules that can bind to target bacteria with high affinity and specificity.Similar to antibodies, aptamers also serve both as the recognizer and linker in the sandwich structure construction.However, they have advantages over the antibodies.Firstly, screening process is both time-saving and cost-effective, known as the systematic evolution of ligands by exponential enrichment (SELEX).It is an in vitro selection process which usually takes 2 to 3 months (the quickest being 2 weeks) in contrast to at least 3 to 6 months required by monoclonal antibody preparation.Secondly, aptamers have better chemical stability than antibodies.Aptamers possess a certain degree of thermal annealing features, and can maintain chemically stable over a wide pH range from 2 to 12, whereas antibodies are easily denatured due to their protein nature.Thirdly, aptamers can be flexibly modified with functional groups because it can be synthesized with a simple and cost-effective chemical method.As a better replacement of antibodies, aptamers have been widely used in bacteria detection, and some even in cancer cells detection.Herein, Ag coated MnFe 2 O 4 (MnFe 2 O 4 @Ag) particles are used as the MPPs, synthesized through the "seed-mediated growth" method and modified with Aptamer-1 as shown in Fig. 11a.A novel SERS-tag, with DTNB-labeled insideand-outside plasmonic NPs, is prepared and modified with aptamers, as shown in Fig. 11b.The sensing principle is similar to that of immuno-assay, but forms an aptamer-targetaptamer sandwich complex, as shown in Fig. 11c.  is plotted with regarding the vibration band at 1331 cm -1 , as shown in Fig. 12b.The calibration line exhibited a good linearity with a correlation coefficient of 0.9558 under the bacteria concentrations ranging from 10 to 10 5 cells/mL.
With the aid of a newly-developed SERS mapping technique, single cell detection of bacteria becomes feasible following the proposed sensing method as shown in Fig. 13.The SEM and optical images of the final MPPs-target-SERS-tag sandwich complex are shown in Fig. 13 (a) and (b), respectively.The SERS mapping image is shown in Fig. 13c.The map color corresponds to the SERS intensity at the vibration band of at 1331 cm -1 .The shape of the mapping image generally depends on the distribution of the SERS-tag that bound on the surface of the bacteria.The SERS spectra acquired from different spots of the sandwich complex are plotted in Fig. 13d.The ability of single cell detection demonstrates the excellent sensitivity of the aptamer recognition sensing method.
Comparing with other recently reported methods, the SERS technique performs better or equivalent well for the bacteria detection, as demonstrated in Table I, 102,[107][108][109][110][111][112][113][114][115] and offers advantages of shorter time and easier operation process.The label-based sensing strategy possesses a better sensitivity than the label-free one, even down to the single cell level, by detecting the spectra of the SERS-tag instead of the instinct spectra of the bacteria.The molecules with good SERS activity are usually selected as the report molecules and immobilized on the SERS-tag.These report molecules are located right at the hotspots area, i.e. at the gap between the MPP and the SERS-tag in the SERS measurements.Thus, the SERS-tag serves as an effective amplifier and transducer in the sensing system, leading to improved sensitivity by two orders of magnitude.Compared with the label-based method, labelfree one is more robust and simpler; albeit lower, the sensitivity of the label-free method is often acceptable in many cases.There advantages make the label-free method a better candidate for rapid and on-site bacteria filtration.In addition, by detecting the intrinsic spectra of the bacteria, the label-free method can be utilized to characterize the physiological state of the bacteria.

VI. CONCLUDING REMARKS
The good physical properties, cost-effective synthesis, ease of control, and the flexible functionizability make the MPPs a versatile platform for the pathogen bacteria cell detection.The inner magnetic core provides MPP with good magnetic responsivity.And the outer noble metal shell provided with excellent plasmonic activity.It is fundamental to cover the magnetic core with a thin and continuous noble metal, as it can give rise to good SERS performance without affecting the magnetic prosperity significantly.By using an external magnet, MPPs can be easily manipulated to facilitate the target separation and enrichment, performing favorably over the commonly used centrifugation process which may lead to unwanted particle aggregation.The adaptable functionalization confers to MPPs an appropriate affinity and selectivity towards target analyte.Taking advantage of the particular chemistry or biology groups at the surface of MPPs, the capture efficiency can be improved and the proposed SERS platform can be expanded over a broad range of biological conditions.
The combination of advanced MPPs and the promising SERS technique has paved the way for sensitive, selective, rapid and cost-effective bacteria sensing.Despite these remarkable developments, however, challenges remain.Systematical studies are required in the immediate future to improve the reproducibility of the detection method, especially for the quantitative analysis, as the SERS signal is highly sensitive to the size, shape and distribution of the particles.In addition, the whole-organism fingerprinting database should be built for the competent bacteria sensing.The database is of particularly importance for the label-free detection.Moreover, optimization of parameters for the sensing systems is still required to meet the demands of clinical diagnostics for the future development of personalized medicine.The fundamental advantages of MPPs have generated a dramatic increase in their use in the bacterial sensing, which will continue to rapidly evolve, along with advances in other fields such as microfluidics, molecular biology, analytical chemistry, and self-assembly techniques.This will open many great opportunities for coordinated international studies and continue to revolutionize the field of diagnostics for years to come.

FIG. 8 .
FIG. 8. (a, c) TEM and SEM images of the MPPs-bacterial aggregation without and with the Au@Ag NPs addition, respectively.(b, d) SERS spectra of E. coli BL21 with different concentrations obtained via the MPPs and CEE method, respectively.

FIG. 9 .
FIG. 9. Synthetic route of the SERS-based immuno-sensing method and TEM images of each step.(a) Synthetic route and (b) TEM image of gold shell-coated magnetic nanoparticles, (c) schematic illustration of the operating procedures for bacteria detection via a SERS method, TEM images of (d) S. aureus binding to antibody-conjugated MPPs and (e) MPPs-bacteria-SERS-tag sandwich architecture.

101 - 106 FIG. 11 .
FIG. 11.Schematic illustration of aptamer recognition S. aureus detection using the SERS method.Synthesis and functionalization of (a) the MPPs and (b) the SERS-tag.(c) the operating principle for S. aureus detection.

FIG. 13 .
FIG. 13.Single-cell detection of S. aureus by SERS mapping method.The corresponding (a) SEM image, (b) optical image, and (c) SERS intensity map of aptamer recognition sandwich architecture.(d) Average SERS spectrum obtained from the different regions of (c).

TABLE I .
The characteristics of recently reported methods for bacteria detection.