Immunoreaction-Based Sensors to ImproveBacterial Detection

Huilin Zhang, Nanjia Zhou, and Feng Ju

ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Basic Mechanisms of Bacterial Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Optical Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Colorimetric Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Fluorescent Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Surface Plasmon Resonance Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Electrical Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Electrochemical Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Piezoelectric Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

The Material and Fabrication of the Biochip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13The Materials of Biochip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13The Fabrication of Biochip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Bacterial Sensors for the Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

H. ZhangDivision of Environment and Resources, School of Engineering, Westlake University, Hangzhou,Chinae-mail: zhanghuilin@westlake.edu.cn

N. ZhouDivision of Nanotechnology and Energy, School of Engineering, Westlake University, Hangzhou,Chinae-mail: zhounanjia@westlake.edu.cn

F. Ju (*)Division of Environment and Resources, School of Engineering, Westlake University, Hangzhou,China

Division of Environment and Resources, Westlake University, Hangzhou, Chinae-mail: jufeng@westlake.edu.cn

© Springer Science+Business Media, LLC, part of Springer Nature 2020M. Sawan (ed.), Handbook of Biochips,https://doi.org/10.1007/978-1-4614-6623-9_38-1

1

Abstract

Bacterial sensors are an emerging interdisciplinary field integrating advances infields of materials science, chemistry, and biology enabling rapid and low-costdetection of bacteria. Optical and electrical biosensors have been under fastdevelopment in recent years for their rapidness and low measurement cost. Thischapter presents the common sensing principles, immunoreaction-based detec-tion methods, and their recent applications in this field. Biochip promotes bacte-rial sensors by integrating them into user-friendly, cost-effective, andminiaturized platform. The main materials and the fabrication of biochip areintroduced. It is advantageous to integrate the biochips and bacterial sensorsinto facile and sensitive detection methods and devices.

Introduction

Harmful bacteria, as one major cause of illness, pose a serious threat to public safety andpeople’s health. Bacterial detection is a key to the prevention and pertinent control of theoutbreaks or aggravation of various diseases, such as tuberculosis, typhoid, cholera,tetanus, leprosy, sepsis, urinary tract infection, food poisoning, etc. The conventionalmethods for bacterial detection mainly include agar plating, polymerase chain reaction,and enzyme-linked immune-sorbent assay. These approaches either require expensiveinstrumentation and highly trained personnel, or they are time-consuming and laborintensive. In the past decades, various biosensors have emerged as alternatives for rapiddetection of harmful and cultivable bacteria from food, animals, human, and environ-ments. The typical targets for biorecognition include the whole bacterial cell and theintracellular proteins and nucleic acids (i.e., DNA and RNA) (Ahmed et al. 2014). Theimproved design of bacterial sensors requires more rapid, on-site applicable, andattractive alternative methods. The application of biochips provides essentially minia-turized and high-throughput platforms for biochemical reactions with minimal require-ment in the volumes of samples and reagents, ideal mixing conditions, and state-of-the-art detection principles and devices (Kumar et al. 2013). Two commercialized examplesof biochips, PhyloChip and GeoChip, are ultra-high-throughput, fast, and low-costDNA microarray technologies developed and optimized to quantify more than thou-sands of bacterial phylogenetic markers and functional genes from numerous environ-mental or humanmicrobial samples in parallel. Therefore, bacterial sensors based on thebiochips exhibit significant advances, appreciable achievements, and promising futurefor the cost-effective bacterial diagnosis in the clinical and environmental settings (Kimet al. 2009; Kumar et al. 2013).

This chapter provides an overview of immunoreaction-based optical sensors andelectrical sensors that are commonly used because of their short detection time andgood data reliability at relatively low costs. Recent advances in the technologies andmonitoring mechanisms related to bacterial detection are briefly introduced. We alsoreview the materials and the fabrications of the biochips, together with some recent

2 H. Zhang et al.

examples of bacterial sensors and their advantages and limitations. Finally, wesummarize perspectives and challenges in the field of bacterial sensors.

Basic Mechanisms of Bacterial Sensors

Bacterial sensors based on biochemical or biological mechanisms are a new type ofbioanalytical method involving multidisciplinary knowledge of information tech-nology, biotechnology, analytical chemistry, materials science, physics, etc. Thebasic mechanisms of such bacterial sensors are shown in Fig. 1. In brief, a biometriccomponent is fixed on a specific transducer, i.e., the system that translates the signalfrom one kind of device to another. The target in the sample biochemically reactswith the biometric component fixed on the transducer by diffusion or active mixing.This step generates electrical, thermal, or optical signals. The target quantity can becalculated according to the signal strength.

In this chapter, the main types of this kind of bacterial sensors including theoptical biosensors and electrical biosensors are described. The significant advantagesand limitations of these biosensors are briefly summarized in Table 1.

Optical Biosensor

Optical biosensors are convenient biosensors that typically utilize light emission orlight absorption for the detection of bacteria and output optical signals as thedetected objects. Usually, the process involves the transition between the energylevels of certain molecules or nanoparticles contained in the sensing element (Banica2012). It is mainly based on absorbance, fluorescence, or reflection to monitorbacterial targets. An optical method is widely applied in field detection because ofits advantages such as being portable, rapid, and cost-effective.

Colorimetric Biosensor

Colorimetric biosensor plays an important role in the detection of bacteria, especially inthe point-of-care diagnostics. These sensors are portable, rapid, and convenient tooperate (Table 1). The user can easily observe the change of color with naked eyeswithout the need for additional analytical instrumentation. Its basic working principle isthat the change of the number (or concentration) of target bacterial cells in the samplecan cause a proportional color change of the detection solution; thus the exact number ofthe target cells in the sample can be predicted and measured according to the change ofthe color. The common substances that can change color and are applied mainly includeenzyme-substrate, precious metal nanoparticles, acid-base indicator, etc.

Tetramethylbenzidine (TMB) is widely used in the chromogenic reaction. It canbe catalyzed and activated by horseradish peroxidase (HRP), and consequently itscolor changes from blue to yellow after stopping the reaction with hydrochloric acid.

Immunoreaction-Based Sensors to Improve Bacterial Detection 3

The color change system of TMB-HRP is a typical methodological implementationin colorimetric biosensors (Fig. 2). The chromogenic reactions are usually coupled

Interference

BiomoleculesTarget

Transduction

Signal

Fig. 1 A schematic of a typical bacterial biosensor

Table 1 Immunoreaction-based bacterial sensors reviewed and their advantages and challenges

Sensor Advantages Challenges Ref.

Colorimetricbiosensor

Convenient operation, fast,handheld device, smartphoneintegratable, and flexible arraysize

Relatively high detection limit,reproducibility of imaging,instability, and short shelf life

[1–2]

Fluorescentbiosensor

Simultaneous detection, highsensitivity, and selectivity

Sensitive to pH and oxygenchange, biocompatibility, andphotostability issues

[3–4]

Surfaceplasmonresonancebiosensor

Label-free, real-time detection,and high sensitivity

High detection limit, bulky insize, sensitive to motion,sweat, and temperature, andlong calibration time

[5–6]

Amperometricbiosensors

High selectivity, sensitivity,low-cost, and continuousdetection

Poor universality, complexitiesfor sensor design, electrodemodification, and operation

[7–8]

Impedancebiosensor

Rapid detection, low detectionlimit, and label-free detection

Prone to noise from ions,selectivity in real samples,reusability, and reproducibility

[9–11]

Piezoelectricbiosensor

High sensitivity, label-freedetection, and microfluidicintegratable

Prone to noise from impurityand high performancepiezoelectric thin films

[12–13]

[1–2] Farka et al. 2018; Kangas et al. 2017; [3–4] Strianese et al. 2012; Xu, Callaway, Wang & Li,2015; [5–6] Taylor, Ladd, Yu, Chen & Jiang, 2007; Villena Gonzales et al. 2019; [7–8] Bahadir andSezginturk 2016; Xu et al. 2016; [9–11] Chen et al. 2015; Daniels and Pourmand 2007; Xu et al.2016; [12–13] Fu et al. 2017; Shen et al. 2011

4 H. Zhang et al.

with a bacterium-specific immunoreaction. The target is captured by a monoclonalantibody which is immobilized on a plate. The HRP is bonded to the polyclonalantibody by the reaction between streptavidin and biotin. The sandwich is formeddue to the antigen-antibody reaction and the HRP-driven chromogenic catalysis ofTMB. Depending on the concentration level of the bacterial target, the final color ofthe substrate will be different. Enzymatic methods are widely used in colorimetricbiosensors for their intuitive results. However, the development of natural enzymestypically involves a high production cost, and their activities often decrease duringchemical reactions. The demand for and application of cheaper mimic enzymes areincreasing. Take Prussian blue as an example, this dye is a kind of mimic enzymecomposed by K4[Fe(CN)6] and K3[Fe(CN)6]. It can also catalyze TMB for itsperoxidase-like activity. Prussian blue can be coated by the treated bull serumalbumin (BSA) and form the sandwich with antibody and target. After adding theTMB, the absorbance can be detected by a spectrometer to achieve the quantitativeanalysis of the target (Farka et al. 2018). With the development of facile electronicplatforms, intelligent recognition shows a favorable advantage in colorimetric anal-ysis, and smartphones have been often used for image collection and the visualizeddetection of targeted bacterial cells. Recently, Zhang et al. developed a bacterialsensor on capillary based on Fe nanoclusters and smartphone imaging (Zhang et al.2019). Based on the principal that the iron ions (Fe3+) could be released from thenanocluster bacteria and react with K4[Fe(CN)6] to form Prussian blue, this systemwas developed for detecting different concentrations of the targets by monitoring the

Target

Monoclonal antibody

Polyclonal antibody

EnzymeHigh con.

Low con.

No target

Color 1

Color 2

Colorless

Fig. 2 A schematic of a colorimetric biosensor based on immunoreaction

Immunoreaction-Based Sensors to Improve Bacterial Detection 5

color intensity of Prussian blue using a smartphone, showing a high sensitivity formonitoring spiked Salmonella spp. in milk.

Fluorescent Biosensor

Fluorescence is a kind of photoluminescence phenomenon. It is generated by amolecule transition from the ground electronic level to a higher level (Banica2012). Fluorescent biosensor exhibits simultaneous detection in transduction; thusit is also widely used in bacteria detection. Usually, the basic working principle is tointroduce the fluorescent substance into the detection system by means of immuno-logical binding, and then the qualitative or quantitative analysis of the target can begained based on the dependent relationship between the fluorescence intensity andthe concentration of the target bacteria.

Quantum dots and fluorescein are the common fluorescent materials used as themeasurable signals in the detection system. The quantum dots with different sizescan emit fluorescence at different wavelengths. In this sense, multicolor quantumdots can be combined with immunomagnetic separation to realize the simultaneousdetection of multiple targeted microorganisms. Xu et al. developed a fluorescentbiosensor that can simultaneously detect four foodborne pathogens, namely,Escherichia coli O157:H7, Staphylococcus aureus, Listeria monocytogenes, andSalmonella typhimurium (Xu et al. 2015). In brief, the immunomagnetic beadswere first modified with different antibodies. The above four pathogens were thenisolated from enriched food samples. After that, the quantum dots of the fourdifferent wavelengths bonded with antibodies were added to form a sandwichstructure. Finally, the quantum dots were detected based on spectrograph, andfluorescence intensity was used to determine the concentration of foodborne patho-gens. Under optimized conditions, the detection limits of these four pathogens inbeef could achieve as low as ~102 CFU/mL within only 2.5 h. As a chemicalmolecule, fluorescein is easily bonded with protein or DNA with stable exposurein a long time. Furthermore, a fluorescent microsphere formed by the polymermicrosphere and the fluorescent molecule is often used to amplify the signal.Recently, Wang et al. combined a fluorescent biosensor with microfluidic technol-ogy. This largely simplified the pretreatment of the sample and organically integratedthe whole detection system (Wang et al. 2019). The targets were labeled withimmune fluorescent microspheres, and a smartphone app that utilizes inter-framedifference algorithm was developed to monitor the concentration of the Salmonellatyphimurium in apple juice (Fig. 3).

Surface Plasmon Resonance Biosensor

Surface plasmon resonance (SPR) is widely used in the detection of bacteria due toits label-free operation, high sensitivity, and compatibility for real-time detection(Table 1). Plasmon is a large group of electrons in the oscillation state. The surface

6 H. Zhang et al.

plasmon is formed when the light irradiates at the surface of certain metals, and thisphenomenon is termed as surface plasmon resonance (Campbell and Xia 2007). Thedetection principle is shown in Fig. 4. Typically, the biological components (anti-body or aptamer) that can recognize the targets are first bonded to the gold surface ofthe crystal. Upon sample addition, the target in the sample selectively binds to thebiometric materials, thereby causing an increase in the mass of the crystal surfaceand, thereafter, a change in the refractive index of the light. Finally, the number oftarget bacteria can be calculated by detecting the change of the light refractive index.

For the detection of bacteria, a SPR chip often needs to be modified withantibodies. It is feasible to achieve the simultaneous detection of multiple types ofbacterial targets by integrating multiple modification reactions with microfluidics.Taylor et al. reported a novel method for the simultaneous and quantitative analysisof four foodborne pathogens by multichanneled SPR biosensors (Taylor et al. 2007).Eight different antibodies were used to modify an eight-channel SPR sensor,followed by injecting apple juice samples containing Escherichia coli, Salmonellaspp., Listeria spp., and Campylobacter spp. Under the optimized conditions, thedetection limit of this method ranges from 3.4 � 103 to 1.2� 105 CFU/mL for thesefour common foodborne pathogens.

Despite its aforementioned advantages, the SPR chip that utilizes reflected lightfrom the signal is susceptible to interference. Researchers have tried to improve thesignal-to-noise ratio by adding the metal nanoparticles on the surface of the SPR chip

Otherimpurities

Magneticseparation

Magneticseparation

Fluorescentmicroscopic system

Outlet

Inlet

LED light source

Salmonella typhimurium MNP FMS MAb PAb

Smartphone App for real-time video processing

Unboundfluorescent

microspheres

Fig. 3 The detection of Salmonella typhimurium based on a fluorescent biosensor integrated withmicrofluidic technology (Wang et al. 2019). The target bacteria were first isolated and then formedthe sandwich with the fluorescent microspheres. The fluorescent spots in the microfluidic chip couldbe monitored using smartphones

Immunoreaction-Based Sensors to Improve Bacterial Detection 7

(Mitchell 2010; Vaisocherova-Lisalova et al. 2016). For example, gold nanoparticleswere used to amplify the signal in the SPR bacterial sensors (Vaisocherova-Lisalovaet al. 2016). In specific, a three-step detection assay was utilized to achieve both therapid and sensitive detection of pathogens based on ultralow fouling andfunctionalized poly(carboxybetaine acrylamide) (Fig. 5). The detection limits forEscherichia coli and Salmonella spp. in hamburger could reach down to 57 and7.4 � 103 CFU/mL, respectively.

Electrical Biosensor

Bacteria can be detected based on the changes in the electric properties of the mass ormedium through measuring changes of electrical properties. In this section, electro-chemical and piezoelectric are introduced.

Electrochemical Biosensors

Electrochemical biosensors usually use electrodes as conversion elements to immo-bilize biometric materials (antibodies, aptamers, enzymes, etc.) on the surface of theelectrodes. The biometric materials specifically separate the target from the complexsample. Then the electrode can convert the biochemical reaction signal into anelectrical signal to achieve the qualitative or quantitative analysis of the target.According to the types of signals, electrochemical biosensors can be divided into

Sensor chip

Prism

Polarized Reflected

Target Antibody Impurities

Fig. 4 A schematic of surface plasmon resonance biosensors modified from Campbell and Xia(2007)

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impedance-, current-, potential-, and conductivity-based biosensors. Due to the lowsensitivity of potential- and conductivity-type biosensors, the electrochemical bio-sensors in current research are mainly designed based on current and impedancesignals for bacterial detection.

Amperometric BiosensorAmperometric biosensors are widely used in the detection of bacteria for their highselectivity, sensitivity, low cost, and continuous detection (Table 1). It can detectbacteria quantitatively by monitoring the change of current. The relationshipbetween electric current and the concentration of the analyte can be expressed bythe Cottrell equation (Grieshaber et al. 2008):

i ¼ nFAC0

ffiffiffiffiffiffi

D j

p

ffiffiffiffiffi

πtp ð1Þ

where i is the current to be tested (A), n is the number of electrons, F¼ 96,485 C/mol(the Faraday constant), A is the area of the electrodes (cm2), C0 is the originalconcentration of analyte (mol/cm3), D is diffusion coefficient of the target (cm2/s),and t is the operation time (s).

Currently, the commonly used methods mainly include cyclic voltammetry, pulsemethod, sine method, and square wave method. There are three kinds of electrodes inthe system including working electrode, reference electrode, and counter electrode.Two peaks (reduction and oxidation) in the current-voltage curve will be generatedwhen the isosceles triangle-shaped pulse voltage is applied to the working electrode.

Fig. 5 A schematic of a three-step assay for the detection of bacterial pathogens in food samples(Vaisocherova-Lisalova et al. 2016). The antibody was first immobilized on the SPR chip and capturedthe bacteria; after washing, the gold nanoparticles were linked to the bacteria by another antibody

Immunoreaction-Based Sensors to Improve Bacterial Detection 9

If the potential of the first half is scanned toward the cathode, the electroactivesubstance is reduced on the electrode to generate a reduction wave. When thepotential of the second half is scanned toward the anode, the reduced product isagain oxidized on the electrode to generate an oxidation wave. Therefore, a trian-gular wave scan completes a cycle of reduction and oxidation processes known ascyclic voltammetry Fig. 6a.

The current-type biosensor measures the concentration of the target microorgan-ism by measuring the current signal and usually generates a current signal by usingenzyme catalysis to generate a redox reaction. A common current biosensor sche-matic is shown in Fig. 6b. It is usually based on the enzyme system (Xu et al. 2017).First, the target in the sample is specifically captured by the antibody on theelectrode, and then an antibody linked to a label such as an enzyme combinedwith the bacteria to form a double-antibody sandwich complex. Finally, the electro-chemically inert substance is catalyzed as an active substance by an enzyme to causea redox reaction. The quantitative detection of the target bacteria can be gained bymeasuring the change in the current signal during redox reaction.

Impedance BiosensorElectrical impedance is the impedance that the circuit shows when an alternatingvoltage is applied. Electrochemical impedance spectroscopy is an advanced methodto study the response of electrochemical systems and monitor the recognitionprocesses. In general, the ohmic resistance of the solution, the capacitance at theelectrode/solution interface, and the rate constant of the electrochemical reaction canall be affected by the identification reaction that occurred at the electrode surface(Banica 2012). The impedance biosensor is designed to provide detectable values foronly the ohmic resistance. At present, a study has reported the use of impedancebiosensors for detecting foodborne pathogenic bacteria (Yang et al. 2004). The basicworking principle based on the immunoreaction and the use of K3[Fe(CN)6] andK4[Fe(CN)6] as a redox pair is shown in Fig. 7. First, the antibody is immobilized onthe surface of the microelectrode of an interdigitated electrode array. Prior to surface

Fig. 6 The cyclic voltammogram (a) and the schematic mechanism of an amperometric biosensor(b) (Xu et al. 2017)

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modification, the electron transfer resistance at the electrode surface is small, whichcontributes to small impedance. After surface functionalization, the impedance willincrease with the increase of the electron transfer resistance. Upon the capture ofantigen by antibody, the electron transfer resistance is further increased, and theimpedance value of the electrode is thus further increased. Electron transfer resis-tance has a good linear relationship with E. coli in the concentration range of ~105 to~108 CFU/mL (Yang et al. 2004). The amount of the targets can be eventuallyquantitatively detected by detecting the change of impedance.

Although advantages such as simple instrument, rapid, low-cost, and easy inte-gration facilitate the wide use of impedance biosensors, the complex electrodemodification processes are required during the manufacture of the traditional imped-ance biosensor. For example, the biomolecules need to be immobilized on theelectrode surface. However, when antibodies directly adsorbed into the metal sur-face, their bioaffinity will be reduced, which lead to a low capture ratio of target andunstable biosensor. In recent years, researchers have developed a new type of

Fig. 7 Principle of an impedance biosensor based on immunoreaction (Yang et al. 2004)

Immunoreaction-Based Sensors to Improve Bacterial Detection 11

modification-free impedance biosensor based on magnetic separation and ureasecatalysis which greatly improves sensitivity (Chen et al. 2015; Xu et al. 2016). Theprocesses can be described as follows (Fig. 8): the target bacteria in the sample arefirst captured by immunomagnetic beads modified with a monoclonal antibody; acatalytic enzyme (e.g., urease, glucose, and oxidase) is then added to form a beads-bacterial-enzyme sandwich complex; upon the addition of catalytic substrate (e.g.,urea glucose), the substrate will be catalyzed to generate a large number of ions; andfinally the change in the impedance value can be detected by the electrode.

Piezoelectric Biosensor

Piezoelectric biosensors use piezoelectric materials as sensing materials and monitorthe target by acoustic waves. Different sound waves can be excited in the medium bydifferent transducers, generally including bulk acoustic waves, surface acousticwaves, plate waves, and love waves. Bulk acoustic waves are sound waves with ageneral frequency between 5 and 20 MHz. They propagate inside the medium. Thethinner the medium is, the higher the detection sensitivity would be. Piezoelectricsensors with bulk acoustic waves are also referred to as quartz crystal microbalance(QCM). QCM is widely used in bacterial detection for its high sensitivity and user-friendliness. The microbial targets can be captured by immobilizing biometricmaterials on the surface of the crystals, thereby causing mass changes with whichthe target can be detected by monitoring the resonance frequency changes of thequartz crystals. The relationship between mass and resonant frequency can beexpressed by the Sauerbrey equation below:

ΔF ¼ �2:26F20Δm=A ð2Þ

where ΔF is the natural resonant frequency of quartz wafer (MHz); Δm is the Qualitychange (g); and A is the effective piezoelectric surface area of quartz wafer (cm2).

Substrate

Target

Ions

Electrode

Monoclonal antibody

Polyclonal antibody Enzyme

Immunomagnetic beads

Fig. 8 Principle of an impedance biosensor based on enzymatic catalysis

12 H. Zhang et al.

The constant is inversely proportional to the density (2.648 g�cm�3) and shearmodulus of the quartz wafer. The negative sign indicates that the surface quality ofthe quartz crystal increases, causing the vibration frequency to decrease.

Up until now, most piezoelectric biosensors use a QCM chip for microbialdetection. Shen et al. used immunomagnetic beads to capture target bacteria andthen combined them with streptavidin-modified gold particles to form a magneticbead-bacteria-gold particle complex, which was used to amplify the signal and thenimmobilized in quartz using protein A (Shen et al. 2011). The antibodies coated onthe surface of the crystal specifically captured the target bacterial cells, and, finally,the resonance frequency was measured. This resulted in the detection of Escherichiacoli O157:H7 down to a low detection limit (only 23 CFU/mL) within 4 h.

The Material and Fabrication of the Biochip

Due to the high complexity in the noise background of food or other environmentalsamples, it is usually necessary to separate the target bacteria from the complexsample before the detection of pathogenic bacteria. In this case, the process of thedetection of bacteria often includes sample collection, target separation, detection,and data analysis. The emergence and increasing popularity of microfluidic chipsprovide a remarkable alternative for the traditional detection system. It allows real-time integration of multiple analysis methods and rapid on-site detection on acompact chip. The major materials and the fabrication of the biochip are introducedas follows.

The Materials of Biochip

The materials that are used to fabricate the biochip mainly include polymers,inorganic materials, and biomaterials such as papers. In the past decades, PDMS(Polydimethylsiloxane) has become the most popular choice for the manufacturingof biochips due to its low cost and easy processability. Compared with othermaterials such as polycarbonate, polymethylmethacrylate, etc., PDMS has goodgas permeability which is favorable for the growth of microorganisms. However,the surface of PDMS is hydrophobic and is prone to non-specific gas adsorption. Inthis case, a hydrophilic treatment is required. In addition, it is difficult to processPDMS featuring complex three-dimensional structures. Alternatively, hydrogelmaterials with excellent biocompatibility are also commonly used to fabricate thebiochip. Microfluidic channels made with hydrogels promote the transport anddiffusion of small molecules in the channel. Glass is a typical representative ofbiochip made of inorganic materials. The advantage of glass is that it is chemicallystable, high strength, and good thermally conductive at high temperatures; thusaccurate nanoscale channels can be gain by processing its surface. The concept ofpaper-based microfluidic chips was proposed by the George Whitesides team(Bracher et al. 2009). Because of its low cost, good biocompatibility, and ease of

Immunoreaction-Based Sensors to Improve Bacterial Detection 13

manufacturing, paper chip technology has been rapidly developed, and multiplecomplex functions can be realized on a single paper chip device. However, the limitsof paper chip mainly include the high detection limit, the loss of sample due to theopen channel in the paper chip, and the difficulty in controlling the fluid.

The Fabrication of Biochip

To date, the main methods for manufacturing biochips mainly include photolithog-raphy, soft photolithography, and 3D printing. As a traditional method, photolithog-raphy is capable of processing nanoscale features on different substrates such assilicon and glass. Firstly, the photoresist is spin-coated on the surface of the glassinto a thin layer. The photoresist is exposed to ultraviolet light under the occlusion ofthe abrasive; after the target surface is etched with hydrofluoric acid, the channel ispackaged to another smooth plan. The main disadvantages of photolithography arehigh production costs, time-consuming processing, and the use of hazardousreagents. Soft photolithography is rapid prototyping which can design and fibratechannel using PDMS. In this process, a 3D model is first printed to develop thepattern. Prepolymer is subsequently cast and allowed to cure. Then, this PDMSreplica is removed from the master, oxidized, and sealed. This technology hasgradually matured and can easily realize the fabrication of the chip without expen-sive instruments. However, it is still time-consuming and requires multiple pro-cessing steps. 3D printing is an additive process with high precision. Thanks to itsrapid development, 3D printing is becoming more popular with further reduction ofmaterial and equipment costs. The advantages of 3D printing are simple operationand one-step molding of the chip without a pattern. Furthermore, the materials frompolymers to various types of biological materials all can be selected to meet specificneeds.

Bacterial Sensors for the Future

Bacterial sensors are an interdisciplinary new field combing advances in the field ofmaterials science, chemistry, biology, etc., enabling rapid and cost-effective detec-tion of bacteria with particular medical, clinical, food safety, and environmentalimportance. In the past decades, various biosensors have been developed for user-friendliness, low-cost, and multiplexed detection of biomaterials (e.g., enzymes,antibodies, aptamers, and DNA) closely related to pathogen diagnostics, animalhealthcare, and environmental and food safety. However, several challenges stillremain for their future real-world applications. First, most bacterial sensors need aprior cultivation step before detection due to the high complexity and usually lowabundance of the targets in the samples. This step may, however, favor enrichmentthus detection of those well-known and easy-to-culture bacteria, whereas neglectthus underestimates the majority of bacterial cells which are not cultivable undertoday’s known laboratory conditions. To overcome or reduce such cultivation

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intrinsic effects, priority may be given to developing bacterial sensors with lowerdetection limit to avoid cultivation or with minimal cultivation before robust detec-tion. This effort would also reduce the required detection time thus allowing in thefuture more timely diagnosis of clinically important, multidrug-resistant bacterialpathogens including carbapenem-resistant, ESBL-producing, and/or vancomycin-resistant Enterobacteriaceae.

Another issue lies in the loss of or reduced sensitivity of today’s lab-scalebacterial sensors when applied to practical samples. For example, the immunologicaldetection method is a widely used method for biosensor detection. As this methodrequires the recognition of antigen and antibody, the specificity and sensitivity of thesensor are deeply influenced by the biometric identification. Since the biosensordetection method often involves many experimental steps, the detection process iscumbersome and may result in poor reproducibility of the analytical results.

The future development of bacterial biosensors will focus on several aspects,namely, miniaturization, multifunctionality, intelligence, and smart integration. It ispromising to develop a new generation of low-cost, high-sensitivity, high-throughput (e.g., with multiple targets), and high-stability biosensors combinedwith the new materials and manufacturing processes. It is well expected that bacterialsensors integrated within a biochip for bacterial detection are promising and willbecome more and more significant and popular.

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