Many conventional large-scale synthesis methods suffer from low efficiency of reactant loading and deficiency of flexibility for tuning parameters of the fabrication. Moreover, due to poor control over mixing and nucleation conditions, it is difficult to form reproducible nanoparticles by conventional bulk synthesis. In recent decades, usage of microfluidic systems has created the ability to fabricate nanoparticles with better control of size distribution, improved reproducibility, high encapsulation efficiency, low sample/solution consumption, high surface to volume ratio, efficient mixing and better control of mass and heat transfer [7
]. Generally, the microfluidic technique handles and controls liquid at the microscale; therefore, nanoliter or even smaller volumes [2
] of fluids can be precisely mixed, and the chemical reactions take place in short time and space scales. A comparison of microfluidic and conventional bulk synthesis is presented in Table 1
Microfluidic devices offer several advantages for the production of polymer, polymer composite and metal particles. By using them, the improvement of the particle size, its shape, morphology, composition and size distribution can be achieved. In order to prepare emulsion of a liquid monomer, two different strategies can be used (Figure 1
). In the first strategy, fluids of continuous and dispersed phases flow inside microchannels and both are in direct contact with the device’s wall. Another strategy describes capillary-based devices, where continuous phase flows inside a tube around a dispersed phase, which is applied by a smaller capillary in the way that the diameter of the formed droplets is much smaller than the channel diameter [8
]. In both cases, the device material (hydrophobic for hydrophilic droplets or vice versa) must be carefully selected or modified to reduce a desired wall contact or a phase inversion.
2.1. Preparation of Sensor Particles in Microfluidic Systems
By using the microfluidic technique, various nanoparticles such as organic, metal, oxide nanoparticles and composites of organic–inorganic nanoparticles can be produced in reproducible and controllable manner. Additionally, during microfluidic synthesis, particles surface can be functionalized with different functional groups; hence, particles have unique physical and chemical properties [2
With application of continuous single flow systems for multistep reactions, the agents for reaction can be easily added and precisely controlled. Santamaria’s group [11
] presented a multistep continuous single-phase microfluidic system for synthesis and functionalization of gold nanorods. By using this platform, formation of seeds and different stages of nanorod growth were separately controlled. Due to this, the reproducibility and quality of the particles were improved. Additionally, the amount of poly-(ethylene glycol)-methyl ether thiol (SH-PEG) used for surface functionalization was reduced 100 times in comparison to batch process. In addition, the microfluidic system was used for tuning the concentration of halide (chloride and bromide) ions at various stages of synthesis. Variable ion concentrations influenced the final nanorods’ aspect ratio and accordingly the optical properties of nanorods.
By utilizing continuous-flow systems, nanoparticles can be synthesized with better reaction conditions control, fast heat and mass transfer as well as high reproducibility [9
]. Knauer et al. [12
] presented a segmented flow method for fabrication of noble metal core/shell and multishell nanoparticles in colloidal solutions. The particles were synthesized by reduction of HAuCl4
salts at the surface of seed particles by ascorbic acid. An improvement of the particle quality was achieved due to the efficient reactants mixing in droplets because of internal convection. It led to a narrower size distribution and lower particle diameter in comparison to the particles produced by conventional batch synthesis. The average diameter and distribution half width size were 20 and 3.8 nm for Au/Ag core/shell as well as 45.6 and 7.4 nm for Au/Ag/Au multishell nanoparticles. It was demonstrated that plasmonic resonance shifted effectively due to the formation of core/shell structures. Therefore, prepared core/shell particles could be used for sensing and labelling applications.
Wacker et al. [13
] presented droplet-based polydimethylsiloxane (PDMS) microfluidic chip for synthesis of silica nanoparticles, whose diameter was controlled via reaction time and reagent concentration. Silica nanoparticles were synthesized by the Stöber method and functionalized with fluorescein-isothiocyanate (FITC) fluorescent dye. When compared to a bulk process, it was shown that the growth speed of nanoparticle was higher in the droplet-based synthesis. For that reason, it was possible to control the size of the nanoparticles over a wider range and much faster. As a result, improvement of nanoparticle size uniformity was seen due to 3% relative standard deviation for 350 nm silica nanoparticles. Moreover, photobleaching of functionalized nanoparticles with FITC was slower in comparison to the photobleaching of pure fluorescein isothiocyanate in water. Despite high controllability of nanoparticle size, particle production rate was relatively low.
L. Zhang et al. [9
] demonstrated a promising approach to synthesize silver nanocrystals with controlled size and shape in droplet-based microreactors. During a seed-mediated synthesis, the nanocrystals were formed from seeds in the way that every seed evolved into the nanocrystals. A gas–liquid droplet microreactor was used for droplet generation (Figure 2
). The device consisted of three syringes, which were filled with air, seed solution (Ag seeds and polyvinylpyrrolidone (PVP) in ethylene glycol (EG) solution) and precursor solution (AgNO3
and PVP in EG). The air segments created by using a droplet microreactor were important not only for separating the reaction solution but also for providing O2
, generating reducing agent. Due to this, homogeneous nucleation and unwanted merging of the single seeds were prevented. It was demonstrated that by varying the amount of seeds or the time of the reaction, or AgNO3
concentration in the segments, edge lengths of Ag nanocubes could be tuned from 30 to 100 nm. Additionally, Ag octahedras were synthesized by reducing the concentration of PVP in the segments. However, the limitation of the presented method was associated with limited syringe volume because continuous flow must be interrupted for reagent reloading.
2.2. Polymer and Hydrogel Particles with Embedded Sensor Dyes
Fluorescent nanosized sensors are the most attractive for sensing purposes. Some materials (quantum dots and fluorescent proteins) show intrinsic fluorescence, but more often, NP fluorescence is created as a result of immobilization of fluorescent or phosphorescent dyes. In order to obtain extrinsic fluorescence, the particles need to incorporate one or more fluorophore dyes on its surface or inside the matrix by entrapping physically or via covalent attachment. Fluorescence sensing is based on changes in fluorescence signals due to fluorescence component interaction with an analyte molecule [1
Generally, pH-sensor particles consist of two essential parts: a polymer matrix which has appropriate adhesive properties and a pH-indicator, which can be immobilized inside the particle matrix or on the particle surface. D. Aigner et al. [21
] presented pH-sensors that consisted of 1,4-diketopyrrolo-[3,4-c]pyrroles (DPPs) as a pH-sensitive dye. As the immobilization matrices, different polymer hydrogels were used in order to achieve high sensitivity, brightness and wide operational range from pH 5 to 12. For the preparation of sensor nanoparticles, 100 mg of positively charged acrylate polymer Eudragit RL100 was dissolved in 50 mL of acetone. Later, indicator dye (DPP) and Macrolex Yellow (1.25 mg) were added with 300 mL of water. By rotary evaporator, acetone was removed from the solution and the particle suspension was prepared. Two fluorescence quenching mechanisms were associated with the ability of DPP indicators to change fluorescence properties in different pH media. Due to the deprotonation of phenolic group, the photoinduced electron transfer (PET) dominated from pH 5.9 to 9.3. The deprotonation of DPP core (lactam nitrogen) occurred at higher pH media (9.7–11.6). Unlike the static planar sensors, which were limited to short-time applications, it was possible to use sensor beads dispersed in the sample for applications in microfluidic systems, fluorescence imaging or microscopy.
Another example of fluorescent pH nanosensors was presented by S. M. Borisov et al. [22
]. Sensors were prepared by staining the poly(styrene-block-vinylpyrrolidone) (PS-PVP) beads with pH dyes consisted of fluorescein and 1-hydroxypyrene-3,6,8-trisulfonate derivatives. By changing substituents on the pH indicator, it was possible to modify the pH-sensing properties. Lipophilic fluorescein derivatives were easily immobilized into the PS-PVP particles without any dyes leaching. This made various beads promising for application in biotechnology and many other fields.
For ratiometric pH imaging, M. P. Gashti et al. [14
] reported silver core–silica shell ([email protected]2
) nanoparticles covalently incorporated with fluorescein isothiocyanate (FiTC) fluorophore. The pH-sensitive sensor nanoparticles were made in two steps. First of all, the seed-growth method of reduction for silver nitrate by sodium citrate was applied for formation of metallic cores with an average size of 80 nm. Secondly, tetraethylorthosilicate (TEOS) was co-condensed with a silanized molecule of fluorescein isothiocyanate (FiTC-APS). In this study, fluorescein was used as a pH indicator because its spectroscopic properties (absorption spectra and fluorescence quantum yield) depend on the pH. By varying the pH value, fluorescein can be in protonated or negatively charged forms and, therefore, applied for quantitative pH measurements.
Oxygen sensitive dyes are usually embedded in the polymer matrix in order to form sensor particles or oxygen sensitive layers. Monodispersed fluorescent platinum octaethylporphyrin-polystyrene (PtOEP/PS) spheres for oxygen monitoring were presented by K. Zhang et al. [15
]. Fluorescent PtOEP/PS particles, whose size varied about 251–384 nm, were prepared by a soap-free emulsion polymerization. It was shown that the fluorescence intensity significantly decreased with the increased dissolved oxygen concentration due to fact that PtOEP molecules fluorescence was quenched by dissolved oxygen, which could easily interact with dye because of porous PS particles structure. Moreover, PtOEP/PS sensors were stable, highly sensitive and showed a fast response time and linear dependence on dissolved oxygen concentration. After 3 months of the preparation of PtOEP/PS spheres, their photostability was investigated. PtOEP/PS particles were illuminated for 1 h under irradiation at 380 nm, and fluorescence intensity of PtOEP decreased only 2.2%. Photo-stability of PtOEP embedded in the PS spheres was much better in comparison to PtOEP in THF solution.
Oxygen nanosensor particles PS-PtTPTBP were used by J. Cao et al. [6
] for non-invasive monitoring of oxygen during growth of bacteria. For the preparation of polystyrene (PS) nanoparticles doped with oxygen-sensitive dye PtTPTBP, first PS particles were synthesized from styrene/water mixture and K2
under temperature control. After the dialysis process, a suspension of nanoparticles was dispersed in methanol/water (2:1) solution and then PtTPTBP, dissolved in chloroform, was added. The mixture was shaken for 4 h and afterwards filtrated and dialyzed. Produced particles were 357 ± 53 nm in size.
Core−shell poly(styrene-block-vinylpyrrolidone) (PSPVP) particles were synthesized by J. Ehgartner et al. [23
]. For simultaneous detection of O2
and pH, the oxygen indicator (PtTPTBPF) was embedded in the core and a pH sensitive BF2
-chelated tetraarylazadi-pyrromethene dye (aza-BODIPY) and antenna dye (Zn-Schiff base), which was used for enhancement of brightness, were incorporated into the shell of the particle. Ideally, all dyes should be embedded into a single particle but due to interactions between dyes and their migration in the core and shell, it was not achieved. Therefore, nanoparticles embedded with oxygen and pH sensitive dyes were prepared separately and mixed together for m-DLR measurements.
In addition to pH, oxygen monitoring, the detection of organic molecules and inorganic ions are also very important for chemical analytics. Therefore, fluorescent micro and nanoparticles are widely used for local chemical sensing. For detection of chloride ions, K.P. Kronfeld et al. [16
] synthesized polyacrylamide hydrogel particles loaded with lucigenin dye due to the fact that its fluorescence is quenched by chloride ions. The continuous flow synthesis of hydrogel sensor particles is shown in Figure 3
. For droplets generation, a monomer and photoinitiator mixture was injected into the carrier phase (silicone oil). After the formation, droplets were irradiated under UV light, which initiated a free radical polymerization of hydrogel particles. Particles were produced with narrow size distribution and an average size of 800 μm. After washing and drying, the size of particles decreased by up to 480 μm. Dry particles were added to the aqueous solution of lucigenin for swelling. After the lucigenin was immobilized into the polyacrylamide matrix, particles were dried again and used for chloride ion detection. It was demonstrated that swelling of sensor particles inside a chloride-containing aqueous solution dramatically influenced the fluorescence quantum yield. Due to the chloride ions, fluorescence intensity decreased in the swollen part of the particle.
J.P.Lafleur et al. [17
] reported a microfluidic sensor for the fast detection of environmental contaminants. The heavy metal Hg and the pesticides ziram were detected by using fluorescent gold nanoclusters and particles. First of all, AuNPs were synthesized by the Frens–Turkevich method [24
]. Then AuNPs were embedded with Rhodamine 6G (R6G), whose fluorescence was quenched when dyes molecules were adsorbed on the surface of AuNPs. Additionally, a 180 mm long meandering channel ensured proper mixing conditions. In order to avoid aggregation of AuNP, the optimal quantity of R6G was found and used for preparation of sensor particles.
2.3. Preparation of Composite Particles
There are several motives for assembling particles for sensing. Sensor NPs can be densely packed into a swellable or nanoporous matrix by forming submicron- or micrometer-sized sensor particles. The preparation of metal/polymer micro and nanoparticles for SERS sensing gives a higher sensor signal because a large amount of small metal particles is incorporated in the composite [1
J.M. Köhler et al. [25
] applied a microcontinuous-flow process in order to prepare hydrogel and silver nanoparticle composites. First of all, silver seed particles were formed in microsegmented flow by reducing silver nitrate via sodium borohydride. Then, silver seeds were used to fabricate silver nanoprisms by using ascorbic acid and silver nitrate. Prepared silver nanoprisms were mixed with an acrylamide monomer, cross-linker and photoinitiator, and droplets were generated in micro co-flow arrangement when polydimethyl-siloxan was used as a viscous carrier phase. The composite particles were produced by a photopolymerization step under UV irradiation. By applying microfluidic synthesis, high reproducibility and narrow size distribution of particles were achieved. Furthermore, washed composite particles were mixed with silver nitrate and ascorbic acid solution for nanoparticle-catalyzed silver deposition on the particle surface in order to improve sensitivity for SERS sensing.
Visaveliya et al. [26
] reported the microfluidic assisted formation of nanoassembly particles. In this robust approach, PMMA-polyDADMAC/gold (Au) nanoassemblies were formed under a flow condition. For formation of copolymer nanoparticles, the microflow-through platform was applied (Figure 4
). Therefore, the particle size was tunable in the range of 200 nm–1 μm by varying the flow rate ratio and concentrations of monomer and carrier phases. The formation of nanoassemblies was based on electrostatic adsorption of negatively charged 30 nm size Au nanoparticles were attracted on the surface of the positively charged polymer particle. In order to improve SERS sensing properties, Ag deposition in the presence of silver precursor and reducing was initiated by the adsorbed AuNPs on the composite particle. Formed PMMA-polyDADMAC/Au/Ag composite was used for adenine sensing. Due to the small size, sensor particles could be applied for SERS sensing not only in the batch condition but also in the flow arrangement.
N. Hassan’s group [27
] reported a two-step microfluidic assisted self-assembly formation of plasmonic, fluorescent and magnetic nanostructures. Rhodamine isothiocyanate (RITC) doped silica particles, superparamagnetic γ-Fe2
nanocrystals and gold NPs were self-assembled under continuous flow in the microfluidic system that consisted of two Y-shaped microreactors made from glass (Figure 5
). Due to the fact that the assembly processes are dynamic, the desired structures can be obtained by varying flow rates of fluids and residence time. By comparing the assemblies prepared in microfluidic process and in bulk conditions, it was demonstrated that the lower density of Au NPs on silica nanoparticles surface was found in bulk. As a result, the high potential of microfluidics in nanoparticle synthesis for imaging purposes was achieved.
A second motivation for assembling can be given by combination of sensor signals. E. Scheucher et al. [28
] prepared multifunctional composite particles that combined oxygen sensing, magnetic and luminescence properties. Luminescence-based composites for the oxygen sensing consisted of the matrix material (PPSQ), upconverting nanoparticles (UCLNPs), magnetic nanoparticles (EMG1300), mediator dye (MFR) and a dye with far-red emission (PtTPTBP). All components were swollen and dissolved in chloroform (Figure 6
). After solvent evaporation, the mixture was heated for 4 h at 230 °C temperature. After, the bulk material was grinded in order to achieve the sensor particles. Formed composite particles had a characteristic luminescence upconversion feature. Under 980 nm laser excitation, particles emitted 545 nm light, which was absorbed by the mediator dye (MFR) and then non-radiatively transmitted to the PtTPTBP. The quenching of this indicator dye is strongly dependent on the oxygen concentration. Therefore, it was possible to apply sensor particles for quantitative oxygen detection in gaseous and dissolved form. Magnetic particles enabled another important feature—the ability to locate particles in a certain position and separate sensor particles from the sample by applying an external magnetic field.
The combination of different NPs inside a composite particle can also be useful for labelling purposes. S-P. Chen et al. [29
] presented microfluidic immune assay, which was based on the shell/shell/core structured nanocomposite. For this purpose, super-paramagnetic iron oxide nanoparticles (“SPIONs”) were particularly interesting. The entire preparation procedure of Au/chitosan/Fe3
involved three crucial steps. Firstly, microwave-assisted solvothermal reaction was used for fabrication of magnetic microspheres. Secondly, magnetic microspheres were coated with the chitosan particles in order to design a shell–core structure. Thirdly, solutions of chitosan/Fe3
and Au colloid were mixed together under stirring, and the final shell/shell/core structure was formed. Due to the incorporated magnetic particles (Fe3
), it was possible to handle and control composite sensor particles with external magnetic field. Additionally, assembled colloidal gold nanoparticles created an ability to immobilize a hemoglobin-A1c antibody (HbA1c mAb). Prepared composite nanoparticles with immobilized antibody were used in microfluidic systems for process controlled and low-volume detection of hemoglobin A1c.
2.4. Preparation of Microcapsules
Multifunctionalized polymer microcapsules, which had a monitoring property of intracellular environments, were presented by G. B. Sukhorukov et al. [30
]. Moreover, it was demonstrated that microcapsules can encapsulate a large variety of molecules such as enzymes, proteins and drugs and release encapsulated compounds in a controlled manner upon an external trigger. Functionalization of nano and microcapsules with luminescent cadmium telluride (CdTe) QDs quantum dots gave the ability to facilitate imaging and labelling of various capsules. For the formation of capsule-based pH sensor, pH indicator dye (SNARF-1) was encapsulated via covalent bonding. The knowledge of local pH values gave important information of cell activity for the reason that changes in the pH were associated with numerous enzymatic reactions and many metabolic processes. Additionally, capsules were targeted to desired cells by applying different surface coatings. Incorporation of magnetic Fe3
nanoparticles into capsule shells allowed the control of the microcapsules by applying external magnetic field. It was shown that microcapsules containing embedded magnetic nanoparticles can be transported with a magnet and be carried to a particular place inside a living cell. Then, the microcapsules were activated or opened to release encapsulated molecules by the laser-induced remote, which gave the possibility to deliver compounds into the cell with precise control.
In order to detect proteins and nucleic acids, S. K. Verma et al. [31
] developed a microcapsule-based assay that featured simplicity, selectivity and sensitivity. Four layers of polyallyllamine hydrochloride (PAH) and polyacrylic acid (PAA) were sequential deposited by electrostatic forces on the 6 μm size CaCO3
particle to form the (PAH/PAA)2
microcapsules. 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) was used to covalently cross-link carboxylic groups of PAA to amine groups of PAHs. By coating the surface of microcapsules with protein A or streptavidin, it was possible to further attach a target biomolecule—for example antibodies, biotinylated oligonucleotides or MHC class I proteins. Prepared microcapsules were stable and did not aggregate under storage or experimental conditions. The detection was carried out on the surface of the microcapsule, when the analyte was squeezed in between a binder and a detector molecule. For evaluating the assay’s sensitivity and selectivity, biomarker protein (hβ2
M) and oligonucleotide molecules were used as analytes. As a result, the assay was highly sensitive to hβ2
M in the concentration range from picogram/L to nanogram/L, despite the presence of irrelevant proteins (streptavidin and conalbumin) in the sample. Moreover, it was shown that biotinylated ligands were strongly bound to the streptavidin-coated (PAH/PAA)2
T. A. Kolesnikova et al. [32
] reported a method for enhancing the targeting of cell surface receptors by applying biofunctionalization of polyelectrolyte (PAH/PAA)2
microcapsules. In this study, two antibody immobilization strategies on the (PAH/PAA)2
microcapsule surface were compared (Figure 7
). In the first case, the specific cell-targeting antibodies were immobilized randomly due to direct covalent coupling of the amino groups of an antibody to the carboxyl groups of a microcapsule surface via EDC/sulfo-NHS reaction. In the second case, before antibody immobilization, microcapsules were coated with protein A, which formed a supporting layer for more effective and optimized coupling of antibodies. The cell surface MHC class I receptors were chosen for a selective binding to antibody-functionalized (PAH/PAA)2
microcapsules. It was found that the targeting efficiency of MHC class I receptors increased by 40–50%, when the antibodies were immobilized on the protein A-coated microcapsules. Moreover, used microcapsules revealed a low cytotoxicity and reduced non-specific binding.
2.5. Metal Particles for LSPR-Based Sensing
Surface plasmons resonance (SPR) is a resonant oscillation of the incident light wave with the oscillations of charge density at metal and dielectric media interface [4
]. For metallic rough surfaces, nanoparticles and sharp tips surface plasmons are thought to be confined in the nanoscale location by forming so-called localized surface plasmons resonance (LSPR), whose characteristic absorption band depends on species, size as well as shape of nanoparticles or metallic structures [33
LSPR-based particle sensorics are promising for the detection of changes in the refractive index (RI), which are caused by the analyte binding [1
]. Non-labelling detection is the main advantage of the LSPR sensor compared with other techniques such as an isotope labelling or fluorescence [34
]. Moreover, LSPR-based biosensors have unique properties allowing biomolecular analysis at real-time and detection of antigen–anti-body interaction or DNA hybridization even when the concentration of analyte is very low [35
]. The binding of analytes on the surfaces of Au and Ag nanoparticles causes the changes in the surrounding environment’s refractive index, which affects the spectroscopic properties (spectral shift or change in intensity) of the particle [35
]. However, the LSPR signals can be affected by non-specific binding of molecules. Therefore, a sample for LSPR sensing should be pure and contain as little impurities as possible [34
For practical purposes, colloidal gold or silver nanoparticles are embedded on the surface of solid material, where the laser light is usually used for excitation the LSPR. Then, optical setup collects the light, which is scattered by the sample [36
]. Due to the fact that the plasmonic effects can happen only near the metallic nanostructures, whose sizes are in nanometer range, LPSR-based sensing can be integrated in microfluidic systems [1
]. Examples of particles used in microfluidic systems for LSPR-based sensing are given in Table 3
H. Sadabadi et al. [37
] proposed a sensitive LSPR-based biosensor, which was applied for polypeptide detection. Using the proposed sensing system, the antigen–antibody interaction of bovine growth hormone was detected with the detection limit of 3.7 ng/mL as low as 185 pM; therefore, it was possible to use the biosensor for clinical applications. For LSPR sensing, gold nanoparticles were synthesized in a PDMS chip during an in-situ reaction between a gold precursor and polymer cross-linking agent. Despite the fact that the synthesis of the particles in microfluidic system was slower, formed AuNPs demonstrated 8.3 times better polydiversity of the size distribution. Four steps of the biosensing protocol are shown in Figure 8
. Before performing the experiment, the gold nanoparticles were functionalized with 11-mercaptoundecanoic acid. After attaching a linker, the activation of the carboxyl groups was carried out and after it, the antibody was attached to the surface of the functionalized gold nanoparticle. In the last step, the antigen was attached by introducing the solution of bovine growth hormone into the microfluidic channel. The immobilization of bovine growth hormone onto AuNPs resulted in increased refractive index and changes in the LSPR spectra. However, in order to improve and restore the proposed microfluidic biosensor for further measurement, the device can be integrated with a pump system and flow cells for channel rinsing purposes.
Another microfluidic system for effective refractive index sensing was reported by B. Doherty et al. [38
]. An integrated optofluidic system, which required a low sample volume and allowed a real-time monitoring of analyte, could be applied for non-invasive analysis and molecular diagnostics. Specific binding of molecules was sensed via plasmonic resonance shift of gold nanoparticles, which strongly depended on the changes in the environment’s IR. The efficient RI sensing was performed with a nanoparticle-functionalized suspended core fiber (SCF). The surface of interior channel was modified with amino groups in order to create adhesive layers for self-assembled monolayers of Au nanospheres. The nanoparticle layer deposition (NLD) technique was used to coat Au nanoparticles along the entire fiber channel length. The RI sensitivity measurements of various RI oils showed that, in the presence of the oil, LSPR shifted towards a longer wavelength. It was determined that the refractive index sensitivity of sensor was 170 nm/RIU for an aqueous analyte. In comparison to the sensitivity of similar gold nanoparticles dispersed in water, the proposed nanoparticle-functionalized fiber was 1.5 times more sensitive.
H. Bhardwaj et al. [39
] presented a AuNP-based biosensor chip for Aflatoxin B1 (AFB1) sensing. Colloidal AuNPs were synthesized using the seed-mediated method [40
]. Surface functionalization of AuNPs was modified by the solvent extraction-based ligand exchange method. First of all, CTAB surfactant was partially removed from the aqueous phase of AuNPs to immiscible organic solvent of dichloromethane. Second, complete replacement of CTAB to lipoic acid was achieved through direct chemisorption. For chemical conjugation of AuNPs with cystamine self-assembly modified SPRi chips, carboxylic functional groups from lipoic acid were activated by using EDC-NHS crosslinkers. After the formation of a self-assembled monolayer (SAM), chemically modified AuNPs were grafted over a SAM Au chip surface for 12 h. Unreacted AuNPs were washed out, and further modifications of SPRi chip were performed directly within the flow cell. After the activation of surface carboxylic acid groups, anti-AFB1 antibodies were immobilized. AuNPs/SAM/Au chips were used for AFB1 detection from 0.01 to 50 nM with a limit of detection of 0.003 nM. Immunosensor responded linearly with various AFB1 antigen concentration due to the binding of AFB1 molecules to immobilized anti-AFB1 at the surface of the sensor. AuNPs acted as signal amplifier and provided a larger area for immobilization of anti-AFB1 antibodies. It was shown that the AuNP integrated sensor chip was three times more sensitive than the SAM/Au sensor chip (15.72 RU/nM).
Y. Zhang et al. [35
] reported an integrated platform combining microfluidic chips and an LSPR method for multiplexed and label-free protein analysis. By using a specially designed optical bench, it was possible to measure the extinction spectrum of the sample in the various regions. The different concentrations of Anti-gp41 antibody, biomarker for HIV/AIDS diagnosis, were investigated in the microfluidic chip with immobilized gold nanoparticles. Nonspecific adsorption of biomolecules was prevented by AuNPs modification with PEG-thiol. It was found that with the increase in the target molecule concentration, the LSPR peak intensity also increased. When comparing results with standard enzyme-linked immunosorbent assay (ELISA), it was shown that detection limits of both methods were similar, but a label-free sensing technique was faster and simpler. In this LSPR-based immunoassay investigation, the importance and effect of linker molecule length were investigated. When linker molecules stand up on the surface, they create a certain distance between the investigated molecule and the surface. Glutaraldehyde was used as the linker between the gp41 antigen and the sensor surface. By applying different linkers, the different thicknesses can be produced. As a result, detection sensitivity was enhanced by using linker molecules with longer spacer arms the due to reduced steric hindrance.
J. He et al. [18
] introduced a 7-channel microfluidic device, which contained nanoparticle arrays, for detection of streptavidin. Photolithography was combined with hole-mask colloidal lithography in order to form homogeneous nanoparticle arrays with particle diameters of 130 nm and a height of 30 nm. Among the biggest advantages for using these methods was increased sensitivity over colloidal particles by patterning nanoparticle arrays of more asymmetric dimensions. It was possible to fabricate microfluidic devices with many channels in comparison to commercial SPR devices. In the same study, glass and PDMS plates, which contained 96 spots of nanoparticle arrays, were fabricated for sensing of protein binding. By using the 96-spot plate, it was possible to simultaneously measure the binding curves of six different protein pairs. After specific antibodies were coupled to nanoparticle arrays, the plate was dried in the room temperature and the reference LSPR spectra were measured. Then, varying concentrations of investigated proteins were added into different plate spots. After drying, the LSPR spectrum was measured, and the shift in LSPR wavelength due to binding of protein was determined. As a result, binding curves were generated, and the limit of detection (LOD) values were calculated.
Integration of an LSPR-based sensor and microfluidic chip was presented by C. Huang et al. [41
]. An antigen–antibody (biotin/anti-biotin) interaction system was used to demonstrate the biological sensing by continuously measuring the transmitted light through the surface of a gold nanoparticle-coated sensor. Gold particles with a diameter of 14–43 nm were synthesized by sodium citrate reduction of chloroauric acid (HAuCl4
]. After, silane layer was used as molecular glue for immobilization of gold nanoparticles on the substrates. Further modification with a self-assembled monolayer (SAM) of thiols was performed in order to carry out the biological experiments. After the immobilization of biotin on SAM, the binding of anti-biotin antibody was detected with a detection limit of 270 ng/mL.
J.S. Chen et al. [20
] presented an LSPR-based biosensor for label-free detection of inflammation-related biomarkers (IgG and CRP). In order to produce Au nanostructures, a thin gold film was deposited on the glass slide using the metallic physical vapor evaporation (PVD) coating technique, which was followed by a rapid thermal annealing (RTA) process. After RTA treatment, the color of the fabricated Au nanostructures changed to dark purple as a result of the LSPR effect. Despite simple and rapid fabrication, formed structures featured poor homogeneity and broad size distribution. The biosensor was integrated into an automated four-channel microfluidic system. For detection of antigens, channels of microfluidic device were functionalized with specific antibodies. Various concentrations of pure IgG antigen or CRP antigen solution were introduced into a sensing device, and specific antibody–antigen binding was investigated by measuring the LSPR absorbance spectrum and monitoring LSPR wavelength shift. It was shown that by using presented sensors, it was possible to detect 10 ng/mL concentration of both IgG and CRP antigens only in 60 μL of sample and in 3.5 h assay time.
Gold nanostructures, particularly spherical nanoparticles, are the most commonly used for LSPR-based sensing despite the fact that silver nanoparticles would give a better sensing performance. Gold nanoparticles are characterized by high stability, and even though silver particles show higher scattering efficiency, their long-time stability is relatively low. However, by forming gold–silver core-shell particles, it is possible to improve sensors’ sensitivity and stability [31
Sensing potential of core–shell nanostructures was reported by A. Steinbrück et al. [43
]. In this research, five different compositions of core–shell nanoparticles were presented, and their sensitivities towards changes in refractive index were investigated. The experimental findings were compared to theoretical calculation and accordingly, experimental results were supported by calculation. For preparation of core–shell NPs, first of all, Au nanoparticles, whose average diameters were 13 and 16 nm, were synthesized according to the Turkevich method [44
]. Later, silver was deposited on the gold nanoparticle surface via an electroless deposition process using the solutions of reducing agent and silver salt. Formation of the Ag shell was proven by color change of the solution (from red to yellow). By varying the concentration of silver salt and reducing agent in the system, the desired thickness of silver shell was formed. It was found that Au–Ag core–shell particles with 2–4 nm shell thicknesses showed higher sensitivity than monometallic gold or silver nanoparticles. Therefore, the LSPR-based sensor could be improved by forming Au–Ag core–shell particles with a specific shell thickness. Due to the fact that optimal shell thickness depends on the range in which the refractive index changes, for different media, core–shell nanoparticles must be designed separately. For the regime interesting for biological application, where changes in refractive index varied from 1.33 to 1.37, the optimal thickness of Ag shell should be 4–5 nm on the 13 nm size gold nanoparticle core.
Due to the fact that the shape of the nanostructures influences the refractive index (RI) sensitivity, rough corners, sharp tips and edges enhance the local electrical field and can strongly affect the sensitivity. M. Thiele et al. [45
] reported the synthesis of Au nanocubes for LSPR sensing application by using a PDMS microfluidic chip. Gold nanocubes were formed from growing gold seeds during a metal-catalyzed deposition of gold atoms, which were reduced by sodium borohydride. Growth of nanocubes was stopped and prevention from overgrowing was achieved by using centrifugation. In comparison to batch synthesis, it was found that Au seed particles, prepared using a single-phase or segmented flow microfluidic methods, were more homogenous and smaller. When sensitivity of the 80 nm Au nanoparticles and 78 nm Au nanocubes was compared, the huge effect of edges and corners was shown. Au nanocubes were 2 times more sensitive (with 202 nm/ RIU) then the Au nanoparticles.
T. Wieduwilt et al. [46
] presented an LSPR sensor based on silver–gold nanoprism immobilization on the optical fiber taper. For the investigation, silver nanoprisms with a size of 40 ± 20 nm and thickness of 9 ± 2 nm were formed by combining synthesis of seed particle and nanoprism growth. The silver nanoprisms were reinforced with a thin gold layer to protect the nanoprism edge against etching. In order to reach a higher sensitivity, fiber optical taper needed to be coated with a dense nanoprism monolayer. It was demonstrated, when density of particles was approximately 210 particle/μm2
, that adjacent particles interacted with each other and boosted the sensitivity. By comparing sensitivities of a hybrid plasmonic–photonic system with bulk solution, it was found that by using tapers with densely immobilized gold-reinforced silver nanoprisms, sensor sensitivity up to 900 nm/RIU was achieved due to interparticle interactions. When interparticle distance between nanostructures increased, sensitivity of the sensor decreased to 385 nm/RIU, which was similar to the sensitivity of a bulk solution.
An economical method to produce metal nanoparticles in a microchannel of glass substrates was proposed by Y. Liu et al. [19
]. In this research, lithography and mask deposition techniques were combined in order to fabricate and firmly placed triangular silver NP arrays in line-shape and cross-shape microgrooves. A sensitivity test of an NP embedded microfluidic chip demonstrated that the LSPR feature of the triangular silver NPs was well maintained. Results of the extinction spectrum of air and water showed that the extinction peak shifted from 706.7 to 783.1 nm by changing the medium from air to water. As a result, sensitivity of the sensor was calculated to be 230.8 nm/RIU.