Silver Nanoparticle-Based Assay for the Detection of Immunoglobulin Free Light Chains

materials

Article Silver Nanoparticle-Based Assay for the Detection of Immunoglobulin Free Light Chains

Anna Lizo ´n 1,* , Magdalena Wytrwal-Sarna 2 , Marta Gajewska 2 and Ryszard Dro˙zd˙z 1

1 Department of Medical Diagnostics, Faculty of Farmacy, Jagiellonian University Collegium Medicum, Medyczna 9, 30-688 Kraków, Poland; [email protected] 2 Academic Centre for Materials and Nanotechnology, University of Science and Technology, 30 Kawiory, 30-055 Kraków, Poland; [email protected] (M.W.-S.); [email protected] (M.G.) * Correspondence: [email protected]

Received: 15 July 2019; Accepted: 12 September 2019; Published: 15 September 2019

Abstract: There is a wide spectrum of malignant diseases that are connected with the clonal proliferation of plasma cells, which cause the production of complete immunoglobulins or their fragments (heavy or light immunoglobulin chains). These proteins may accumulate in tissues, leading to end organ damage. The quantitative determination of immunoglobulin free light chains (FLCs) is considered to be the gold standard in the detection and treatment of multiple myeloma (MM) and amyloid light-chain (AL) amyloidosis. In this study, a silver nanoparticle-based diagnostic tool for the quantitation of FLCs is presented. The optimal test conditions were achieved when a metal nanoparticle (MNP) was covered with 10 particles of an antibody and conjugated by 5–50 protein antigen particles (FLCs). The formation of the second antigen protein corona was accompanied by noticeable changes in the surface plasmon resonance spectra of the silver nanoparticles (AgNPs), which coincided with an increase of the hydrodynamic diameter and increase in the zeta potential, as demonstrated by dynamic light scattering (DLS). A decrease of repulsion forces and the formation of antigen–antibody bridges resulted in the agglutination of AgNPs, as demonstrated by transmission electron microscopy and the direct formation of AgNP aggregates. Antigen-conjugated AgNPs clusters were also found by direct observation using green laser light scattering. The parameters of the speciﬁc immunochemical aggregation process consistent with the sizes of AgNPs and the protein particles that coat them were conﬁrmed by four physical methods, yielding complementary data concerning a clinically useful AgNPs aggregation test.

Keywords: multiple myeloma; amyloidosis; laser light scattering; silver nanoparticles

1. Introduction Multiple myeloma, the second most common hematologic malignancy, is characterized by the clonal proliferation of plasma cells, their prolonged survival, and the accumulation of clonal plasma cells in bone marrow. It is accompanied by the presence of monoclonal immunoglobulin and immunoglobulin free light chains (FLCs) in the serum, urine, or both. FLCs or their deposits may accumulate in tissues, leading to end organ damage. The most common clinical manifestations of symptomatic multiple myeloma are anemia, infections, lytic or osteopenic bone disease, and renal failure [1,2]. Another disease associated with immunoglobulin free light chains is amyloidosis. It is caused by an aggregation of misfolded FLCs or their fragments in vital organs (the kidneys, heart, liver, or peripheral nerves). Deposits of amyloid ﬁbrils lead to an impaired function of aﬀected organs. There is a common consensus that amyloidosis is underdiagnosed. Without an accurate diagnosis and proper treatment, the typical survival period of patients with undiagnosed amyloidosis and cardiac

Materials 2019, 12, 2981; doi:10.3390/ma12182981 www.mdpi.com/journal/materials Materials 2019, 12, 2981 2 of 14 involvement is estimated to be about six months, so a fast diagnosis is crucial. The treatments currently available significantly increase survival [3,4]. From the discovery of the Bence-Jones protein, which was later found to be an immunoglobulin free light chain, a new era in the diagnosis and monitoring of MM and related disorders was ushered in. The quantitative determination of free light chains is considered to be the gold standard in the detection and treatment of multiple myeloma and AL amyloidosis [2,5,6]. Metal nanoparticles (MNPs) are widely used in many fields of medicine, such as diagnostics, therapy, and medical imaging. Silver and gold nanoparticles have attracted a lot of attention due to their unique optical properties, resulting from localized surface plasmon resonance (LSPR) [7,8]. LSRP depends on several factors, such as the size and shape of particles and the distance between them. When the distance between nanoparticles decreases, or there are changes in the dielectric constant of the local environment on the surfaces of MNPs (changes in the nanoparticle environment or particle agglomeration), a change in the absorbance spectrum occurs, which is accompanied by a change in the colloid color [9]. One of the promising uses of MNPs, based on their unique physical properties, is in the development of laboratory assays for detecting different analytes. There are many potential mechanisms for the detection of the biological markers of diseases. One of the mechanisms is the immunochemical interaction between biomolecules of interest and MNPs, which is captured using antibodies. Antibody–antigen interactions are based on biospecific recognition, which has a high selectivity [10,11]. Antibody-antigen interaction can either cause direct agglutination [12,13] or inhibit aggregation caused by another destabilizing factor affecting the optical properties of nanoparticles [14,15]. Nanoparticle-based assays are considered a promising alternative to classic latex assays. Silver and gold nanoparticles (NPs) have a remarkably high absorption coefficient and strongly distance-dependent optical properties. The interaction of antibodies immobilized on MNP surfaces with their antigens causes nanoparticle aggregation and an LSPR shift, which is indicated by a change in a solution’s color [11,16]. While there are many theoretical papers concerning silver nanoparticles’ properties and their possible use in diagnostics, there are still few practical laboratory assays, and the proposed solutions differ significantly. In this pilot study, a diagnostic tool for the detection of immunoglobulin free light chains in urine, based on silver nanoparticles, is presented. E and optimization of the molar ratio of the metal nanoparticles, antibodies, and antigen at which the optical changes occur were performed.

2. Materials and Methods Anti-lambda bound and free polyclonal antibodies (SCE239M) were obtained from Biameditek sp. z o.o. (Białystok, Poland). Free light chain lyophilizate was obtained from urine samples of patients diagnosed with monoclonal gammopathy and Bence-Jones proteinuria. To isolate the protein (FLCs), the salting out method using an ammonium sulfate solution was used. The use of human biological material for the research was approved by the Bioethics Committee of the Jagiellonian University: KBET/161/B/2009 and 1072.6120.248.2017 (25.10.2018). AgNO3, NaBH4, trisodium citrate, and all other chemical reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

2.1. Synthesis of Silver Nanoparticles AgNPs were prepared according to the method reported by Creighton [17]. This method is based on the reduction of silver nitrate using sodium borohydride. Brieﬂy: 0.5 mL of an aqueous solution of AgNO3 (0.01 M) and 0.5 mL trisodium citrate (0.01 M) were added to 19 mL of chilled distilled H2O. Then, a freshly prepared aqueous solution of NaBH4 (0.6 mL, 0.01 M) was added drop-by-drop under vigorous stirring at 0 ◦C. After NaBH4 was added, the vigorous magnetic stirring was maintained for Materials 2019, 12, 2981 3 of 14

3 min. The resulting solution changed to a stable light yellow color, indicating the formation of Ag nanoparticles. The stable silver colloid solution was stored at 4 ◦C until use.

2.2. Functionalization of AgNPs with Antibodies The silver nanoparticles were coated with antibodies by adding 1 µL of the anti-FLCs antiserum to 10 mL of the AgNPs solution. After stirring well, the mixture was incubated for 30 min at 37 ◦C.

2.3. The Detection of FLCs by the AgNPs-ab Probe In order to follow the immunological reaction between antibody-functionalized AgNPs and the target proteins, immunoglobulin free light chains (FLCs), a stock PBS solution, at a concentration of 2.2 g/L, was prepared. The preparation of the primary ab-functionalized AgNPs and antigen system was as follows: working solutions were obtained by mixing the antibody-functionalized nanoparticles (1 mL) and diﬀerent volumes of the stock FLCs solution (1, 2, 3, 4, 5, 7 and 10 µL). The final FLCs concentrations were 2.2–22 mg/L (100–1000 Nm). To obtain lower FLCs concentrations (0.2–1.1 mg/L, 10 nM–50 nM), appropriate dilutions (10 , 8 , 5 , 4 , 3 , 2 ) of the stock solution were added to 1 mL of antibody-coated × × × × × × silver nanoparticles. After stirring them well, the mixtures were incubated at 37 ◦C.

2.4. The Detection of FLCs by the AgNPs-ab Probe

2.4.1. UV/Vis Spectroscopy Citrate-stabilized AgNPs, antibody-modiﬁed AgNPs and AgNPs + ab + FLCs aggregates were analyzed by UV/Vis spectroscopy (MultiscanSky, Thermo Scientiﬁc, Waltham, MA, USA) within a range of 350–600 nm. Path length adjustment to 1 cm was performed.

2.4.2. Transmission Electron Microscopy Transmission electron microscopy (TEM) observations were carried out on a FEI Tecnai TF20 X-TWIN (FEG) microscope (Thermo Scientiﬁc, Waltham, MA, USA), working at an accelerating voltage of 200 kV. Samples for TEM observations were prepared by drop-casting on carbon-coated copper TEM grids. DeltaOptical DLT CamViewer software (ver. x64, 3.7.12277.20180703) was used for TEM image analysis. Diameter values obtained by the distance selection tool previously calibrated to the scale bar imprinted on the TEM images were determined.

2.4.3. Dynamic Light Scattering A Malvern Nano ZS light scattering apparatus (Malvern Instruments Ltd., Malvern, UK) was used for dynamic light scattering (DLS) measurements. The time-dependent autocorrelation function of the photocurrent was acquired every 10 s, with 15 acquisitions for each run. The samples were illuminated by a 633 nm laser, and the intensity of the light, scattered at an angle of 173◦, was measured by an avalanche photodiode. The ζ-averaged hydrodynamic mean diameters (dz), polydispersity (PDI), and distribution proﬁles of the samples were calculated using the software provided by the manufacturer. The following samples were measured: 1) citrate-stabilized AgNPs, 2) AgNPs coated with an antibody, 3) AgNPs coated with an antibody and incubated with FLCs (1 mL:3 µL). The third sample was measured after 30 min, 1 h, and 2 h of incubation at 37 ◦C, with gentle and continuous stirring.

2.4.4. Laser Light Scattering Observations of the laser light scattering patterns of the tested NPs solutions were performed using a self-made measurement system. The system (Figure1) consisted of:

(1) A green semiconductor laser of 532 nm, which was situated above the sample; Materials 2019, 12, 2981 4 of 14

(2) A optical microscope placed horizontally, with 40 magniﬁcation and a long working distance × objective lens placed in the revolver; Materials 2019, 12, x FOR PEER REVIEW 4 of 15 (3) Samples were placed in standard plastic cuvettes (1 cm pathlength); and (4) Scattering patterns were recorded using a SONY alpha 6000 camera (Minato, Tokio, Japan). Materials 2019, 12, x FOR PEER REVIEW 4 of 15

Figure 1. Self-made measurement system for the observation of laser light scattering.

3. Results and Discussion Figure 1. Self-made measurement system for the observation of laser light scattering. 3.1. CharacteristicsFigure 1. Self-made of Silver Nanoparticles measurement system for the observation of laser light scattering. 3. Results3. ResultsPrepared and and Discussion Discussion AgNPs were characterized using TEM, UV/Vis spectrophotometry (Figure 2 and Figure 3), and DLS measurement (Figure 4). The UV/Vis spectrum shows that the resulting AgNPs 3.1.3.1. Characteristicssolution Characteristics has a ofcharacteristic Silverof Silver Nanoparticles Nanoparticles absorption maximum at 395 nm. As the maximum of the plasmon resonance spectrum and diameter of AgNPs are correlated, a maximum of 395 nm indicates the PreparedPrepared AgNPs AgNPs were were characterized characterized using using TEM, TEM, UV UV/Vis/Vis spectrophotometry spectrophotometry (Figures(Figure 2 andand3 ), occurrence of nanoparticles, with dimensions of around 10 nm, which is in accordance with the andFigure DLS measurement 3), and DLS measurement (Figure4). The (Figure UV /Vis 4). spectrumThe UV/Vis shows spectrum that theshows resulting that the AgNPs resulting solution AgNPs has a literature data [18]. solution has a characteristic absorption maximum at 395 nm. As the maximum of the plasmon characteristicThe data absorption were confirmed maximum by at direct 395 nm.determination As the maximum of AgNPs’ of thediameter, plasmon obtained resonance from spectrumTEM resonance spectrum and diameter of AgNPs are correlated, a maximum of 395 nm indicates the and diameterimages. The of AgNPsmean TEM-based are correlated, diameter a maximum of AgNPs ofwas 395 9.62 nm nm indicates (median the9.2 occurrencenm, SD 3.4 nm). of nanoparticles, Figure 3 withoccurrence dimensionsshows the ofTEM ofnanoparticles, particle around size 10 nm,distributionwith which dimensions isofin AgNPs. accordance of around with10 nm, the which literature is in dataaccordance [18]. with the literature data [18]. The data were confirmed by direct determination of AgNPs’ diameter, obtained from TEM images. The mean TEM-based diameter of AgNPs was 9.62 nm (median 9.2 nm, SD 3.4 nm). Figure 3 shows the TEM particle size distribution of AgNPs.

FigureFigure 2. Silver 2. Silver nanoparticles nanoparticles prepared prepared byby aa chemicalchemical reduction reduction of of silver silver nitrate nitrate (V) (V)using using sodium sodium borohydride:borohydride: (a) characteristic (a) characteristic UV-Vis UV-Vis spectrum, spectrum, with with the the maximum maximum absorbance at at 395 395 nm, nm, (b ()b color) color of the solution,of the solution, (c) transmission (c) transmission electron electron microscope microscope image image of of the the synthesized synthesized AgNPs.AgNPs.

Figure 2. Silver nanoparticles prepared by a chemical reduction of silver nitrate (V) using sodium borohydride: (a) characteristic UV-Vis spectrum, with the maximum absorbance at 395 nm, (b) color of the solution, (c) transmission electron microscope image of the synthesized AgNPs.

Materials 2019, 12, x FOR PEER REVIEW 5 of 15

Materials 2019, 12, 2981 5 of 14 Materials 2019, 12, x FOR PEER REVIEW 5 of 15

Figure 3. TEM particle size distribution of AgNPs.

Dynamic light scattering analysis was performed in order to evaluate the physical characterization of silver nanoparticles (Figure 4). The mean diameter of AgNPs, calculated by intensity distribution, was 30 ± 6 nm. The mean and median values of the diameter of the AgNPs, obtained from DLS, were higher than those obtained from TEM. This may be explained by the interference with the hydrodynamic diameter of AgNPs caused by the dispersant. The DLS method measures the mean hydrodynamic diameter, which is heavily weighted toward the largest structures in the solution, while TEM shows particles’ diameter without this aqueous and ionic cargo. Thus, DLS showed a larger mean particle size. Similar observations were described in the study of Souza et al. [19]. As the particle size distribution is not narrow, the presence of bigger particles may contribute to an increase in light scattering, which increases the size of the measured particles. FigureFigure 3.3. TEMTEM particleparticle sizesize distributiondistribution ofof AgNPs.AgNPs.

Figure 4. Dynamic light scattering size distribution (by intensity) curves for AgNPs (black), AgNPs + ab Figure 4. Dynamic light scattering size distribution (by intensity) curves for AgNPs (black), AgNPs + (green), and AgNPs + ab + FLCs (red) solutions. ab (green), and AgNPs + ab + FLCs (red) solutions. The data were conﬁrmed by direct determination of AgNPs’ diameter, obtained from TEM images. 3.2. Preparation of ab-Modified AgNPs for the Detection of FLCs The mean TEM-based diameter of AgNPs was 9.62 nm (median 9.2 nm, SD 3.4 nm). Figure3 shows the TEMFor the particle detection size distributionof protein antigens, of AgNPs. either mono- or polyclonal antibodies can be used. In the case ofDynamic immunoglobulin light scattering free light analysis chains, was which performed display in order significant to evaluate heterogeneity, the physical it characterizationis possible that of silver nanoparticles (Figure4). The mean diameter of AgNPs, calculated by intensity distribution, was 30 6 nm. The mean and median values of the diameter of the AgNPs, obtained from DLS, ± were higher than those obtained from TEM. This may be explained by the interference with the hydrodynamic diameter of AgNPs caused by the dispersant. The DLS method measures the mean hydrodynamic diameter, which is heavily weighted toward the largest structures in the solution, while TEM shows particles’ diameter without this aqueous and ionic cargo. Thus, DLS showed a larger Figure 4. Dynamic light scattering size distribution (by intensity) curves for AgNPs (black), AgNPs + mean particle size. Similar observations were described in the study of Souza et al. [19]. As the particle ab (green), and AgNPs + ab + FLCs (red) solutions. size distribution is not narrow, the presence of bigger particles may contribute to an increase in light scattering,3.2. Preparation which of increasesab-Modified the Ag sizeNPs of for the the measured Detection particles. of FLCs For the detection of protein antigens, either mono- or polyclonal antibodies can be used. In the case of immunoglobulin free light chains, which display significant heterogeneity, it is possible that

Materials 2019, 12, 2981 6 of 14

3.2. Preparation of ab-Modiﬁed AgNPs for the Detection of FLCs

MaterialsFor 2019 the, 12 detection, x FOR PEER of proteinREVIEW antigens, either mono- or polyclonal antibodies can be used. In6 of the 15 case of immunoglobulin free light chains, which display significant heterogeneity, it is possible that the monoclonalthe monoclonal antibody antibody will will not detectnot detect a particular a particular epitope. epitope. However, However, it is it di isffi difficultcult to characterize to characterize the commerciallythe commercially available available polyclonal polyclonal antibodies. antibodies. From a technicalFrom a pointtechnical of view, point polyclonal of view, antibodies polyclonal can consistantibodies of a wholecan consist serum of or a purified whole serum immunoglobulin or purified fraction. immunoglobulin To characterize fraction. the compositionTo characterize and the concentrationcomposition and of the the antibodies concentration used of in the this antibodies study, capillary used in electrophoresis this study, capillary was performed electrophoresis (Figure was5). Theperformed electrophoretic (Figure pattern5). The demonstrated electrophoretic that patt the anti-FLCsern demonstrated serum was that composed the anti-FLCs of a purified serum gamma was globulincomposed fraction. of a purified The concentration gamma globulin of the fraction. immunoglobulin The concentration stock solution, of the immunoglobulin estimated through stock a comparisonsolution, estimated with the through known a concentration comparison with of a the protein known standard concentration (using the of a capillary protein standard electrophoretic (using method),the capillary was 168electrophoretic g/L. This corresponds method), to was 1.2 mM,168 assumingg/L. This a corresponds molecular immunoglobulin to 1.2 mM, assuming IgG weight a ofmolecular 140 kDa. immunoglobulin As the silver nanoparticles IgG weight were of 140 coated kDa. with As antibodiesthe silver nanoparticles by adding 1 µ Lwere of the coated anti-FLCs with antiserumantibodies (stockby adding solution) 1 μL of to the 10 mLanti-FLCs of the AgNPsantiserum solution, (stock thesolution) final concentration to 10 mL of the of AgNPs antibodies solution, was 120the nM.final concentration of antibodies was 120 nM.

Figure 5.5. Capillary electropherogram of of the anti-FLC anti-FLCss serum (blue line) and human serum, as aa referencereference (red(red line).line).

One ofof the the most most important important steps steps in designing in designing an immunochemical an immunochemical test is totest determine is to determine the absolute the concentrationabsolute concentration of all elements of all andelements the optimal and the antibody optimal/antigen antibody/antigen molar ratio molar in order ratio to excludein order the to possibilityexclude the of possibility an antigen of excess an antigen (the hook excess eff (theect). hook effect). The nextnext problemproblem thatthat mustmust bebe solvedsolved isis thethe determinationdetermination ofof thethe antibodyantibody/nanoparticle/nanoparticle ratioratio aaffectingffecting proteinprotein corona corona formation. formation. This This is is a complicateda complicated process process that that depends depends on theon electrostaticthe electrostatic and hydrophobicand hydrophobic interactions interactions between between specific specific antibodies antibodies and aand nanoparticle’s a nanoparticle’s surface surface [9,20 ].[9,20]. Literature datadata onon the optimal number of antibodiesantibodies that should be used for the functionalization of nanoparticles are very very divergent. divergent. There There are are huge huge discrepancies discrepancies concerning concerning the the optimal optimal number number of ofantibodies antibodies that that should should be be added. added. Sometimes, Sometimes, the the data data even even differ differ by by an an order order of of magnitude. Additionally,Additionally, thethe authorsauthors presentpresent didifferentfferent concentrationsconcentrations ofof nanoparticlesnanoparticles inin solutionssolutions andand numbersnumbers of addedadded antibodies.antibodies. Some authors present the concentrationconcentration of AgNPs in milligrams of elementary silversilver/liters/liters [[12,15,21].12,15,21]. To clarifyclarify thethe interpretationinterpretation ofof thethe resultsresults obtainedobtained in this study,study, allall ofof thethe concentrationconcentration datadata werewere presentedpresented in molsin /liters.mols/liters. This approach This providesapproach information provides concerning information AgNPs /antibodiesconcerning/ antigenAgNPs/antibodies/antigen ratios. ratios. In this study, the molar concentration of the solution of AgNPs was estimated, on the basis of the LSPR maximum and the molar extinction coefficient (5.56 × 108 M–1 cm–1), to be 10 nM. The concentration of antibodies, after the functionalization process, was 120 nM. This means that the AgNPs/antibodies ratio was about 10. With this number of antibodies, the nanoparticles are stable, and they do not aggregate spontaneously.

Materials 2019, 12, 2981 7 of 14

In this study, the molar concentration of the solution of AgNPs was estimated, on the basis 8 1 1 of the LSPR maximum and the molar extinction coeﬃcient (5.56 10 M− cm− ), to be 10 nM. Materials 2019, 12, x FOR PEER REVIEW × 7 of 15 The concentration of antibodies, after the functionalization process, was 120 nM. This means that the AgNPsTo/antibodies characterize ratio the was antibody about 10.functionalization With this number process, of antibodies, UV-Vis LSPR the nanoparticles spectra were are recorded. stable, andCompared they do to not citrate-stabilized aggregate spontaneously. AgNPs, antibody functionalization results in a slight red shift and a decreaseTo characterize in the absorption the antibody maximum, functionalization without peak broadening, process, UV-Vis which LSPR is in agreement spectra were with recorded. previous Comparedstudies [22] to (Figure citrate-stabilized 6). The LSPR AgNPs, spectrum antibody right shift functionalization indicates the resultsformation in aof slight a protein red shift corona. and a decrease in the absorption maximum, without peak broadening, which is in agreement with previous studies [22] (Figure6). The LSPR spectrum right shift indicates the formation of a protein corona.

FigureFigure 6. 6.UV-Vis UV-Vis absorption absorption spectra spectra and and TEM TEM image: image:( a(a)) silver silver nanoparticles nanoparticles (AgNPs) (AgNPs) and and ( b(b)) silver silver nanoparticlesnanoparticles after after coating coating with with antibodies antibodies (AgNPs (AgN+Psab). + Theab). TEMThe imageTEM imag highlightse highlights the protein the coronaprotein surroundingcorona surrounding the individual the individu grainsal of grains nanoparticles. of nanoparticles.

TheThe DLSDLS measurementmeasurement showsshows thatthat thethe meanmean diameterdiameter ofof thethe AgNPs-ab,AgNPs-ab, calculatedcalculated byby intensityintensity distribution,distribution, isis 46.146.1 ± 4.1 nm (Figure (Figure 44).). The The typical typical dimensions, dimensions, reported reported in in the the literature, literature, of ± ofimmunoglobulins immunoglobulins are are approximately approximately 14.5 14.5 nm nm × 8.58.5 nm nm × 44 nm nm [23]. [23]. The The presented DLSDLS datadata suggestsuggest × × thatthat immunoglobulins immunoglobulins formed formed a monolayera monolayer on on the the surface surface of AgNPs of AgNPs during during the functionalization the functionalization step. Additionally,step. Additionally, the binding the binding site of thesite immunoglobulins of the immunoglobulins on the silver on the nanoparticle silver nanoparticle surface seemssurface to seems be in theto be constant in the constant Fc domain, Fc domain, leaving the leaving two antigen-recognitionthe two antigen-recognition Fab regions Fab available regions available for antigen for binding. antigen binding.The zeta potential of the modiﬁed AgNPs is higher than that of the citrate-stabilized AgNPs ( 27.3The mV zeta vs. potential36.6 mV), of andthe modified theoretically, AgNPs they is should higher be than more that prone of the to citrate-stabilized aggregation. While AgNPs the − − Zeta(−27.3 potential mV vs −36.6 increased, mV), and the theoretically, antibody-coated they AgNPsshould be were more stable prone in to the aggregation. solution. ThisWhile conﬁrms the Zeta previouspotential observationsincreased, the that antibody-coated the coverage AgNPs of nanoparticles were stable within the proteins solution. increases This confirms their stability.previous Dueobservations to protein–nanoparticle that the coverage hydrophobic of nanoparticles and ionic with interactions, proteins increases protein-coated their stability. nanoparticles Due to protein– do not aggregatenanoparticle [24 ].hydrophobic and ionic interactions, protein-coated nanoparticles do not aggregate [24].

3.3.3.3. DetectionDetection ofof FLCsFLCs UsingUsing ab-Functionalizedab-Functionalized AgNPsAgNPs TheThe detectiondetection ofof FLCsFLCs inin thethe probeprobe isis basedbased onon thethe interactioninteraction betweenbetween speciﬁcspecific antibody-antibody- functionalizedfunctionalized AgNPs AgNPs and and neoplastic neoplastic cell-derived cell-derived protein protein antigens antigens (FLCs) (FLCs) (Figure (Figure7). 7).

MaterialsMaterials2019 2019, 12, 12, 2981, x FOR PEER REVIEW 88 ofof 14 15 Materials 2019, 12, x FOR PEER REVIEW 8 of 15

Figure 7. The mechanism of antigen detection. Specific antibodies were attached to the citrate- Figurestabilized 7.7.The The AgNPs. mechanism mechanism A further of antigenof antigenaddition detection. detection. of the Speciﬁc prot Specificein antibodies antigens antibodies were (FLCs)attached were caus attacheded to thethe citrate-stabilizednanoparticles to the citrate- to AgNPs.stabilizedaggregate. A furtherAgNPs. addition A further of theaddition protein of antigens the prot (FLCs)ein antigens caused (FLCs) the nanoparticles caused the to nanoparticles aggregate. to aggregate. TheThe addition addition of of immunoglobulin immunoglobulin free free light light chains chains to ab-functionalizedto ab-functionalized AgNPs AgNPs is accompanied is accompanied by spectacularby spectacularThe addition changes changes of inimmunoglobulin the in absorptionthe absorption spectrafree spectra light related chai relatedns to to LSPR. toab-functionalized LSPR. These These spectral spec AgNPstral changes changes is areaccompanied relatedare related to thebyto spectacularthe aggregation aggregation changes process process in of the AgNPs,of absorptionAgNPs, which which spectra can can be bere observedlated observed to LSPR. by by TEM, TEM, These DLS DLS spec measurements, measurements,tral changes are and and related direct direct observationstoobservations the aggregation using using process laser laser light light of AgNPs, scattering. scattering. which can be observed by TEM, DLS measurements, and direct observationsIncreasingIncreasing using concentrations concentrations laser light scattering. of of FLCs FLCs are are accompanied accompanied by by a decreasea decrease in thein the maximum maximum absorption absorption at 395at 395 nm,Increasing nm, broadening, broadening, concentrations and red-shiftand red-shift of ofFLCs the of LSPRare the accomp LSPR spectrum. spectrum.anied Additionally, by a Additionally, decrease the in formation the the maximum formation of a second absorptionof a second peak, atpeak, a395 wavelength nm,at a wavelengthbroadening, of around of and around 470 red-shift nm, 470 was nm,of the observed. was LSPR observed. spectrum. Spectral Spectral changesAdditionally, changes that that occur the occurformation after after the additionofthe a additionsecond of dipeak,ofﬀ erentdifferent at a numbers wavelength numbers of immunoglobulin ofof aroundimmunoglobulin 470 nm, free was lightfree observed. light chains chains to Spectral antibody-coated to antibody-coated changes that nanoparticles occur nanoparticles after (withthe addition (with 24 h of24 incubation)ofh differentof incubation) arenumbers presented are presentedof immunoglobulin in Figure in Figure8. 8.free light chains to antibody-coated nanoparticles (with 24 h of incubation) are presented in Figure 8.

FigureFigure 8. 8. SpectralSpectral changeschanges of the nanoparticle aggregates aggregates after after the the addition addition of of different diﬀerent numbers numbers of ofFigureimmunoglobulin immunoglobulin 8. Spectral changesfree free light light of thechains chains nanoparticle to to antibody antibody-coated aggregates-coated afternanoparticles the addition [10 of nM].nM].different TheThe numbers ﬁnalfinal FLCsFLCs of concentrationsimmunoglobulinconcentrations were were free within within light a a rangechains range of ofto 0.5–10 0.5–10 antibody mg mg/L/L-coated (25–500 (25–500 nanoparticles nM). nM). [10 nM]. The final FLCs concentrations were within a range of 0.5–10 mg/L (25–500 nM).

Materials 2019, 12, x FOR PEER REVIEW 9 of 15 Materials 2019, 12, 2981 9 of 14 Concentrations of FLCs (0.5–10 mg/L [25–500 nM]) in the presence of antibody-functionalized AgNPs results in a decrease in absorbance at 395 nm. Further increasing the FLCs concentrations resultsConcentrations in regrowth of of the FLCs absorbance (0.5–10 mg and/L su [25–500bsequent nM]) narrowing in the presence of the absorbance of antibody-functionalized peak. AgNPsFigure results 9 shows in a decrease the changes in absorbance in the absorbance at 395 nm. (at Further395 nm) increasingof antibody-coated the FLCs AgNPs concentrations (10 nM) resultswith increasing in regrowth FLCs of the concentrations. absorbance and The subsequent maximal narrowingabsorbance of changes the absorbance were obtained peak. when the FLCs/AgNPsFigure9 shows molar theratio changes was 50. in Further the absorbance increasing (at the 395 FLCs nm) concentrations of antibody-coated results AgNPs in a regrowth (10 nM) of withthe absorbance. increasing FLCsThis can concentrations. be explained on The the maximal basis of absorbancethe antibody–antigen changes were hook obtainedeffect. when the FLCs/AgNPs molar ratio was 50. Further increasing the FLCs concentrations results in a regrowth of the absorbance. This can be explained on the basis of the antibody–antigen hook eﬀect.

FigureFigure 9. 9.Changes Changes inin thethe absorbance absorbance ofof antibody-coated antibody-coated nanoparticlesnanoparticles (10(10 nM),nM),with withthe the maximum maximum LSPRLSPR (395(395 nm)nm) andand increasing increasing FLCs FLCs concentrations. concentrations. TheThe biggestbiggest changeschanges werewere observedobserved atat aaFLCs FLCs concentrationconcentration of of 10 10 mg mg/L/L (500 (500 nM). nM).

SpectralSpectral changeschanges werewere accompanied accompanied by by a a change change in in the the color color of of the the solution. solution.When Whenthe thesamples samples werewere centrifuged, centrifuged, sedimentation sedimentation of of the the AgNPs AgNPs in in thethe samples,samples, withwith thethe optimaloptimal concentrationconcentration ranges,ranges, tooktook place place (Figure (Figure 10 10).). The largest spectral changes occurred at FLCs concentrations of up to 10 mg/L. At higher concentrations (above 20 mg/L), the reaction was blocked, most likely by a protein corona that forms as a consequence of excess protein (antigen). The range of concentrations at which the reaction may occur indicates the minimal clinically useful sensitivity. This silver NP-based assay suﬀers from typical immunochemical characteristics, resulting from the antigen excess, i.e., the so-called hook eﬀect. In practice, as in other immunochemical tests, the only way to solve this problem is by diluting the sample at higher antigen concentrations [25].

Materials 2019, 12, 2981 10 of 14 Materials 2019, 12, x FOR PEER REVIEW 10 of 15

FigureFigure 10. 10. (a()a )Color Color changes changes of of the the AgNPs AgNPs ++ abab + +FLCsFLCs solu solutions,tions, with with FLCs FLCs concentrations concentrations from from 0 0to to 2222 mg/L, mg/L, which which are are represented by thethe lettersletters a–n.a–n. (b(b)) TEM TEM image image of of silver silver nanoparticle nanoparticle aggregates aggregates (k). (k).Changes Changes in thein the absorbance absorbance of antibody-coatedof antibody-coated nanoparticles nanoparticles (10 (10 nM), nM), with with the the maximum maximum LSPR LSPR (395 (395nm) nm) and increasingand increasing FLCs concentrations.FLCs concentrations. The biggest The changesbiggest werechanges observed were atobserved a FLCs concentration at a FLCs concentrationof 10 mg/L (500 of 10 nM). mg/L (500 nM). 3.4. Detection of FLCs Using ab-Functionalized AgNPs The largest spectral changes occurred at FLCs concentrations of up to 10 mg/L. At higher concentrationsTo further (above characterize 20 mg/L), the the FLCs reaction measurement was blocked, assay most using likely AgNPs, by a theprotein kinetics corona of thethat reaction forms aswas a consequence measured at of the excess selected protein FLCs (antigen). concentration: 7 mg/L (300 nM) (Figure 11). TheThe range biggest of concentrations changes in the at which LSPR the spectra reacti occuron may within occur theindicates first 2the h minimal (Figure clinically11 inset). usefulThe immunochemical sensitivity. This silver reaction NP-based was divided assay intosuffers two from phases. typical During immunochemical the first 2 h, a characteristics, decrease in the resultingmaximum from absorption the antigen at 395 nmexcess, was observed.i.e., the Then,so-called in the hook second effect. phase, In the practice, LSPR absorbance as in other peak immunochemicalbroadens, and ared-shift tests, the and only second way to peak solve at this a wavelength problem is of by around diluting 470 the nm sample appears at higher (Figure antigen 11)[26 ]. concentrationsThis aggregate [25]. formation-induced red-shift in the nanoparticle LSPR resonance spectra has also been well documented in relation to other metal colloids. Similar changes in the absorbance spectra 3.4.during Detection the reaction of FLCs Using between ab-Functionalized the antibody-functionalized AgNPs gold NPs were described in the clinically important immunochemical assay for the detection of glycated hemoglobin [27]. To further characterize the FLCs measurement assay using AgNPs, the kinetics of the reaction This aggregation behavior, found by UV/Vis spectroscopy, was confirmed by DLS measurements was measured at the selected FLCs concentration: 7 mg/L (300 nM) (Figure 11). of the hydrodynamic diameters and zeta potential of antibody-functionalized AgNPs. As the immune The biggest changes in the LSPR spectra occur within the first 2 h (Figure 11 inset). The reaction progressed, an increase in the hydrodynamic diameter of the objects and an increase in the immunochemical reaction was divided into two phases. During the first 2 h, a decrease in the zeta potential were observed. maximum absorption at 395 nm was observed. Then, in the second phase, the LSPR absorbance peak As the immune reaction progresses and protein particles of the measured antigens (FLCs) broadens, and a red-shift and second peak at a wavelength of around 470 nm appears (Figure 11) selectively interact with specific antibodies, the hydrodynamic diameter and zeta potential of the [26]. aggregates increase (measured 30, 60, and 120 min from the beginning of the reaction). An increase in the zeta potential promotes the aggregation of ab-coated AgNPs (Figure 12). After 60 min, the second stage of the reaction is visible, as indicated by the decrease in the hydrodynamic diameter and zeta potential of the aggregating nanoparticles, which is consistent with the formation of a distinct set of absorbance spectra (Figure 11).

MaterialsMaterials 20192019,, 1212,, x 2981 FOR PEER REVIEW 1111 of of 15 14

Figure 11. Kinetics of the immunological reaction, with the selected number of FLCs (7 mg/L). FigureCharacteristic 11. Kinetics changes of the in theimmunological absorption spectrum:reaction, with up to the the selected 2nd h, anumber decrease of inFLCs the maximum(7 mg/L). Characteristicabsorbance at changes 395 nm occurred;in the absorption and later, spectrum: a widening up of to the the spectrum 2nd h, a towards decrease longer in the wavelengths maximum absorbance(red-shift) and at 395 the formationnm occurred; of a second and later, peak a atwideni 470 nmng was of observed.the spectrum The towards inset presents longer time-dependent wavelengths (red-shift)absorbance and changes the formation at 395 nm. of The a second biggest peak absorbance at 470 changesnm was wereobserved. observed The duringinset presents the ﬁrst time- 2 h of Materials 2019, 12, x FOR PEER REVIEW 12 of 15 dependentthe reaction. absorbance changes at 395 nm. The biggest absorbance changes were observed during the first 2 h of the reaction.

This aggregate formation-induced red-shift in the nanoparticle LSPR resonance spectra has also been well documented in relation to other metal colloids. Similar changes in the absorbance spectra during the reaction between the antibody-functionalized gold NPs were described in the clinically important immunochemical assay for the detection of glycated hemoglobin [27]. This aggregation behavior, found by UV/Vis spectroscopy, was confirmed by DLS measurements of the hydrodynamic diameters and zeta potential of antibody-functionalized AgNPs. As the immune reaction progressed, an increase in the hydrodynamic diameter of the objects and an increase in the zeta potential were observed. As the immune reaction progresses and protein particles of the measured antigens (FLCs) selectively interact with specific antibodies, the hydrodynamic diameter and zeta potential of the aggregates increase (measured 30, 60, and 120 min from the beginning of the reaction). An increase in the zeta potential promotes the aggregation of ab-coated AgNPs (Figure 12). After 60 min, the FigureFigure 12.12.Changes Changes in in the the hydrodynamic hydrodynamic diameter diameter (A) and(A) zetaand potential zeta potential (B) of ( a()B the) of citrate-stabilized (a) the citrate- second stage of the reaction is visible, as indicated by the decrease in the hydrodynamic diameter AgNPsstabilized (AgNPs), AgNPs (b )(AgNPs), antibody-functionalized (b) antibody-functionalized AgNPs (AgNPs AgNPs+ ab) and (AgNPs (c) silver + nanoparticlesab) and (c) coatedsilver and zeta potential of the aggregating nanoparticles, which is consistent with the formation of a withnanoparticles antibodies, coated after with the addition antibodies, of FLCs after (AgNPs the addition+ ab +of FLCs).FLCs (AgNPs + ab + FLCs). distinct set of absorbance spectra (Figure 11). 3.5. Investigation of Antibody-Functionalized Nanoparticle Aggregation by Direct Observation Using Laser Light3.5. Investigation Scattering of Antibody-Functionalized Nanoparticle Aggregation by Direct Observation Using Laser Light Scattering The aggregation of antibody-coated silver nanoparticles was also examined by analyzing the scatteringThe aggregation of monochromatic of antibody-coated green (532 nm) silver semiconductor nanoparticles laser was light. also Inexamined the measurement by analyzing system the constructedscattering of for monochromatic the purposes of green the present (532 nm) research, semico variousnductor patterns laser light. of the In light the scatteringmeasurement of uncoated system constructed for the purposes of the present research, various patterns of the light scattering of uncoated nanoparticles, nanoparticles coated with antibodies, and silver nanoparticle–protein aggregates were demonstrated. A narrow beam of laser light, passing through the solution of unfunctionalized silver nanoparticles, gave a homogenous scattering pattern (Figure 13). The functionalization of the nanoparticles with antibodies caused the formation of small light scattering clusters. The addition of protein antigens (FLCs) resulted in the formation of intense light scattering aggregates, which can easily be recognized by analyzing the light dispersion patterns. The obtained scattering patterns were consistent with the results obtained by transmission electron microscopy and sedimentation observations. The data from the light scattering measurements may be used for the qualitative real- time investigation of the protein integration process. The light scattering patterns correlate with the diameter of AgNPs, which can be further quantitated by image analysis software. This method has the potential to offer high sensitivity, enabling the observation of the aggregation of single nanoparticles [28]. The results obtained by DLS demonstrated an increased mean hydrodynamic diameter of nanoparticles (62.8 nm vs. 30 nm for citrate-stabilized AgNPs). However, the DLS method does not show the formation of discrete clusters.

Materials 2019, 12, 2981 12 of 14 nanoparticles, nanoparticles coated with antibodies, and silver nanoparticle–protein aggregates were demonstrated. A narrow beam of laser light, passing through the solution of unfunctionalized silver nanoparticles, gave a homogenous scattering pattern (Figure 13). The functionalization of the nanoparticles with antibodies caused the formation of small light scattering clusters. The addition of protein antigens (FLCs) resulted in the formation of intense light scattering aggregates, which can easily be recognized by analyzing the light dispersion patterns. The obtained scattering patterns were consistent with the results obtained by transmission electron microscopy and sedimentation observations. The data from the light scattering measurements may be used for the qualitative real-time investigation of the protein integration process. The light scattering patterns correlate with the diameter of AgNPs, which can be further quantitated by image analysis software. This method has the potential to oﬀer high sensitivity, enabling the observation of the aggregation of single nanoparticles [28]. The results obtained by DLS demonstrated an increased mean hydrodynamic diameter of nanoparticles (62.8 nm vs. 30 nm for Materialscitrate-stabilized 2019, 12, x FOR AgNPs). PEER REVIEW However, the DLS method does not show the formation of discrete clusters.13 of 15

Figure 13. ScatteringScattering patterns patterns of of monochromatic monochromatic green green semiconductor semiconductor laser laser light, light, passing passing through: through: (a) citrate-stabilized silver nanoparticles (AgNPs), (b) antibody-functionalizedantibody-functionalized silver nanoparticles c (AgNPs + ab),ab), and and ( (c)) nanoparticle nanoparticle aggregates aggregates (AgNPs (AgNPs + abab ++ FLCs).FLCs). The standard methods used for monitoring the immunoglobulin-/antigen-related aggregation The standard methods used for monitoring the immunoglobulin-/antigen-related aggregation of nanoparticles—UV/VIS spectrophotometry, TEM imaging, and dynamic light scattering (DLS)— of nanoparticles—UV/VIS spectrophotometry, TEM imaging, and dynamic light scattering (DLS)— generally provide consistent results. However, each of these methods has some limitations, which generally provide consistent results. However, each of these methods has some limitations, which are due to the method used for preparing a sample for the study or the research methodology itself, are due to the method used for preparing a sample for the study or the research methodology itself, among other factors. Diﬀerent scattering patterns visible in the microscope observations coincide with among other factors. Different scattering patterns visible in the microscope observations coincide the results obtained by other methods. with the results obtained by other methods. The pattern scattering method can be a cheap, relatively simple research method for studying The pattern scattering method can be a cheap, relatively simple research method for studying protein interactions using silver nanoparticles. protein interactions using silver nanoparticles. During the creation of protein corona aggregation research systems, a lot of elements related to During the creation of protein corona aggregation research systems, a lot of elements related to the physical and chemical properties of nanoparticles and protein ligands should be considered. the physical and chemical properties of nanoparticles and protein ligands should be considered. When choosing appropriate imaging methods, nanoparticle-based technology may be useful for When choosing appropriate imaging methods, nanoparticle-based technology may be useful for tracking other reactions related to protein aggregation, e.g., in the study of amyloidosis and other tracking other reactions related to protein aggregation, e.g., in the study of amyloidosis and other diseases associated with the formation of protein deposits. diseases associated with the formation of protein deposits.

4. Conclusions This paper provides an example of the use of silver nanoparticles in the study of protein interactions. The study was focused on a specific, disease-related protein antigen. The obtained model can be practically used to construct an immunochemical test, with a high diagnostic potential. During the development of the method, an appropriate method of synthesizing and functionalizing nanoparticles should be selected to obtain structures with the desired physicochemical properties. The effect of the immunological reaction between silver nanoparticles coated with anti-lambda free light chain antibodies and the target antigen, free light chains of immunoglobulins, were absorbance changes in the area of the maximum surface plasmon resonance, depending on the FLCs concentration. In another nanomolar concentration range of FLCs (25–500 nM; 0.5–10 mg/L), changes in absorbance were accompanied by the aggregation of nanostructures. This is the range of clinically useful concentrations, applicable in the diagnosis of monoclonal gammopathy. It is therefore possible to use the created system to construct a diagnostic test for the quantitative determination of free light chains of immunoglobulins. Similar tests have a key application in the diagnosis and monitoring of monoclonal gammopathy. It is also expected that this novel method can be used in the detection of other serum or urine proteins.

Materials 2019, 12, 2981 13 of 14

4. Conclusions This paper provides an example of the use of silver nanoparticles in the study of protein interactions. The study was focused on a specific, disease-related protein antigen. The obtained model can be practically used to construct an immunochemical test, with a high diagnostic potential. During the development of the method, an appropriate method of synthesizing and functionalizing nanoparticles should be selected to obtain structures with the desired physicochemical properties. The effect of the immunological reaction between silver nanoparticles coated with anti-lambda free light chain antibodies and the target antigen, free light chains of immunoglobulins, were absorbance changes in the area of the maximum surface plasmon resonance, depending on the FLCs concentration. In another nanomolar concentration range of FLCs (25–500 nM; 0.5–10 mg/L), changes in absorbance were accompanied by the aggregation of nanostructures. This is the range of clinically useful concentrations, applicable in the diagnosis of monoclonal gammopathy. It is therefore possible to use the created system to construct a diagnostic test for the quantitative determination of free light chains of immunoglobulins. Similar tests have a key application in the diagnosis and monitoring of monoclonal gammopathy. It is also expected that this novel method can be used in the detection of other serum or urine proteins. Different methods for studying the aggregation of nanoparticles provide consistent, method-specific, and complementary results. The method based on laser light scattering, which was developed in this study, enables the direct observation of the aggregation of single nanoparticles in real time. It combines all of the observations obtained by other methods: UV-Vis spectrophotometry, transmission electron microscopy, and dynamic light scattering. It can be a relatively simple qualitative research method for studying the protein interactions in biological systems using silver nanoparticles.

Author Contributions: Conceptualization, A.L. and R.D.; methodology, A.L., R.D., M.G., and M.W.-S.; DLS and TEM measurements, M.G. and M.W.-S.; manuscript preparation, A.L. and R.D. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

References

1. Kazandjian, D.; Branch, L.M. Multiple Myeloma Epidemiology and Survival, a Unique Malignancy. Semin. Oncol. 2017, 43, 676–681. [CrossRef][PubMed] 2. Dicato, M.A. Multiple Myeloma. Side Eﬀ. Med. Cancer Ther. Prev. Treat. Second Ed. 2018, 385, 277–284. [CrossRef] 3. Vaxman, I.; Gertz, M. Recent Advances in the Diagnosis, Risk Stratiﬁcation, and Management of Systemic Light-Chain Amyloidosis. Acta Haematol. 2019, 55905, 93–106. [CrossRef][PubMed] 4. Poshusta, T.L.; Katoh, N.; Gertz, M.A.; Dispenzieri, A.; Ramirez-Alvarado, M. Thermal Stability Threshold for Amyloid Formation in Light Chain Amyloidosis. Int. J. Mol. Sci. 2013, 14, 22604–22617. [CrossRef] [PubMed] 5. Rock, L.; Hospital, P.; Leukemia, T.; Program, M.; Alfred, R.P.; Wales, N.S.; Higashi, C.; Hospital, G.; Centre, P.C. SPOTLIGHT REVIEW International Myeloma Working Group Guidelines for Serum-Free Light Chain Analysis in Multiple Myeloma and Related Disorders. Leukemia 2009, 23, 215–224. [CrossRef] 6. Bhole, M.V.; Sadler, R.; Ramasamy, K. Serum-Free Light-Chain Assay: Clinical Utility and Limitations. Ann. Clin. Biochem. 2014, 51, 528–542. [CrossRef][PubMed] 7. Egan, J.G.; Drossis, N.; Ebralidze, I.I.; Fruehwald, H.M.; Laschuk, N.O.; Poisson, J.; De Haan, H.W.; Zenkina, O.V. Hemoglobin-Driven Iron-Directed Assembly of Gold Nanoparticles. RSC Adv. 2018, 8, 15675–15686. [CrossRef] 8. Savas, S.; Ersoy, A.; Gulmez, Y.; Kilic, S.; Levent, B.; Altintas, Z. Nanoparticle Enhanced Antibody and DNA Biosensors for Sensitive Detection of Salmonella. Materials 2018, 11, 1541. [CrossRef] 9. Sapsford, K.E.; Algar, W.R.; Berti, L.; Gemmill, K.B.; Casey, B.J.; Oh, E.; Stewart, M.H.; Medintz, I.L. Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries That Facilitate Nanotechnology. Chem. Rev. 2013, 113, 1904–2074. [CrossRef] Materials 2019, 12, 2981 14 of 14

10. Su, H.; Wang, Y.; Gu, Y.; Bowman, L.; Zhao, J.; Ding, M. Potential Applications and Human Biosafety of Nanomaterials Used in Nanomedicine. J. Appl. Toxicol. 2017.[CrossRef] 11. Ajay Piriya, V.S.; Joseph, P.; Kiruba Daniel, S.C.G.; Lakshmanan, S.; Kinoshita, T.; Sivakumar, M. Colorimetric Sensors for Rapid Detection of Various Analytes. Mater. Sci. Eng. C 2017, 78, 1231–1245. [CrossRef][PubMed] 12. Nietzold, C.; Lisdat, F. Fast Protein Detection Using Absorption Properties of Gold Nanoparticles. Analyst 2012, 137, 2821. [CrossRef][PubMed] 13. Xu, X.; Ying, Y.; Li, Y. One-Step and Label-Free Detection of Alpha-Fetoprotein Based on Aggregation of Gold Nanorods. Sens. Actuators B Chem. 2012, 175, 194–200. [CrossRef] 14. Yuan, Y.; Zhang, J.; Zhang, H.; Yang, X. Silver Nanoparticle Based Label-Free Colorimetric Immunosensor for Rapid Detection of Neurogenin 1. Analyst 2012, 137, 496–501. [CrossRef][PubMed] 15. Batistela, D.M.; Stevani, C.V.; Freire, R.S. Immunoassay for Human IgG Using Antibody-Functionalized Silver Nanoparticles. Anal. Sci. 2017, 33, 1111–1114. [CrossRef][PubMed] 16. Sepúlveda, B.; Angelomé, P.C.; Lechuga, L.M.; Liz-Marzán, L.M. LSPR-Based Nanobiosensors. Nano Today 2009, 4, 244–251. [CrossRef] 17. Blatchford, G.; Grant, M. Plasma Resonance Enhancement of Raman Scattering by Pyridine Adsorbed. J. Chem. Soc. Faraday Trans. 2 Mol. Chem. Phys. 1978, 75, 790–798. 18. Zhang, W.; Qiao, X.; Chen, Q.; Cai, Y.; Chen, H. The Influence of Synthesis Condition and Aging Process of Silver Nanocrystals on the Formation of Silver Nanorods. Appl. Surf. Sci. 2012, 258, 5909–5913. [CrossRef] 19. Souza, T.G.F.; Ciminelli, V.S.T.; Mohallem, N.D.S. A Comparison of TEM and DLS Methods to Characterize Size Distribution of Ceramic Nanoparticles. J. Phys. Conf. Ser. 2016, 733.[CrossRef] 20. Tauran, Y.; Brioude, A.; Coleman, A.W.; Rhimi, M.; Kim, B. Molecular Recognition by Gold, Silver and Copper Nanoparticles. World J. Biol. Chem. 2013, 4, 35–63. [CrossRef][PubMed] 21. Wang, X.; Li, Y.; Wang, H.; Fu, Q.; Peng, J.; Wang, Y.; Du, J.; Zhou, Y.; Zhan, L. Gold Nanorod-Based Localized Surface Plasmon Resonance Biosensor for Sensitive Detection of Hepatitis B Virus in Buffer, Blood Serum and Plasma. Biosens. Bioelectron. 2010, 26, 404–410. [CrossRef][PubMed] 22. Lai, W.; Wang, Q.; Li, L.; Hu, Z.; Chen, J.; Fang, Q. Interaction of Gold and Silver Nanoparticles with Human Plasma: Analysis of Protein Corona Reveals Specific Binding Patterns. Colloids Surf. B Biointerfaces 2017, 152, 317–325. [CrossRef][PubMed] 23. Matea, C.T.; Mocan, T.; Zaharie, F.; Iancu, C.; Mocan, L. A Novel Immunoglobulin G Monolayer Silver Bio-Nanocomposite. Chem. Cent. J. 2015, 9, 1–7. [CrossRef][PubMed] 24. Durán, N.; Silveira, C.P.; Durán, M.; Martinez, D.S.T. Silver Nanoparticle Protein Corona and Toxicity: A Mini-Review. J. Nanobiotechnol. 2015, 13, 1–17. [CrossRef][PubMed] 25. Jacobs, J.F.M.; De Kat Angelino, C.M.; Brouwers, H.M.L.M.; Croockewit, S.A.; Joosten, I.; Van Der Molen, R.G. Evaluation of a New Free Light Chain ELISA Assay: Bringing Coherence with Electrophoretic Methods. Clin. Chem. Lab. Med. 2018, 56, 312–322. [CrossRef] 26. Hirsch, L.R.; Jackson, J.B.; Lee, A.; Halas, N.J.; West, J.L. A Whole Blood Immunoassay Using Gold Nanoshells. Anal. Chem. 2003, 75, 2377–2381. [CrossRef] 27. Wangoo, N.; Kaushal, J.; Bhasin, K.K.; Mehta, S.K.; Suri, C.R. Zeta Potential Based Colorimetric Immunoassay for the Direct Detection of Diabetic Marker HbA1c Using Gold Nanoprobes. Chem. Commun. 2010, 46, 5755. [CrossRef] 28. Wang, F.; Chen, B.; Yan, B.; Yin, Y.; Hu, L.; Liang, Y.; Song, M.; Jiang, G. Scattered Light Imaging Enables Real-Time Monitoring of Label-Free Nanoparticles and Fluorescent Biomolecules in Live Cells. J. Am. Chem. Soc. 2019, 141, 14043–14047. [CrossRef]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).