Research Article www.small-journal.com An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays Emilia Renzi, Andrew Piper, Flavia Nastri, Arben Merkoçi,* and Angela Lombardi* sensitive and specific. In this sense, LFAs Signal amplification strategies are widely used for improving the sensitivity of satisfy all the criteria required for ideal lateral flow immunoassays (LFiAs). Herein, the artificial miniaturized per- point-of-care devices (POCDs).[1–3] When oxidase Fe(III)-MimochromeVI*a (FeMC6*a), immobilized on gold nanopar- antibodies are exploited as biorecognition ticles (AuNPs), is used as a strategy to obtain catalytic signal amplification elements, LFAs are categorized as “lateral in sandwich immunoassays on lateral flow strips. The assay scheme uses flow immunoassays (LFiAs)”. LFiAs are AuNPs decorated with the mini-peroxidase FeMC6*a and anti-human-IgG manufactured as strips with four com- ponent parts: the sample pad, where the as a detection antibody (dAb), for the detection of human-IgG, as a model sample solution is added to the strip; the analyte. Recognition of the analyte by the capture and detection antibodies conjugate pad, where the detection anti- is first evidenced by the appearance of a red color in the test line (TL), due to bodies (dAbs), amongst other things, are the accumulation of AuNPs. Subsequent addition of 3,3′,5,5′-tetramethylben- stored; a nitrocellulose membrane, onto zidine (TMB) induces an increase of the test line color, due to the TMB being which antibodies are printed to create a test line (TL) and a control line (CL); an converted into an insoluble colored product, catalyzed by FeMC6*a. This absorbent pad, which enhances the flow work shows that FeMC6*a acts as an efficient catalyst in paper, increasing and adsorbs the excess solution flowing the sensitivity of an LFiA up to four times with respect to a conventional LFiA. laterally along the strip.[3] The pads need Furthermore, FeMC6*a achieves lower limits of detection that are found in to be in direct contact with each other to control experiments where it is replaced with horseradish peroxidase (HRP), allow the sample solution to flow through its natural counterpart. This study represents a significant proof-of-concept them, driven solely by capillary force until wicking.[4] When the output of the assay for the development of more sensitive LFiAs, for different analytes, based on is a colorimetric signal, nanoparticles are properly designed artificial metalloenzymes. often used (because of their high surface- to-volume ratio) to concentrate multiple copies of the dAbs on a small surface.[5,6] 1. Introduction At the time of writing, gold nanoparticles (AuNPs) are com- monly used for this application. AuNPs are popular because Paper-based biosensors, in particular lateral flow assays (LFAs), they offer a variety of surface functionalization protocols, can are commonly used around the world since they are inex- be easily and reproducibly synthesized in a variety of shapes pensive, fast, simple to use and interpret, robust, reliable, and sizes, are colloidally stable, and produce a strong red color (when 20 nm in diameter, as it is most commonly used) which E. Renzi, F. Nastri, A. Lombardi is ideal for the naked eye detection.[7,8] The working principle of Department of Chemical Sciences an LFiA is exhaustively reported in the literature.[4,9,10] In brief, University of Napoli Federico II considering an immune-sandwich assay, when the sample solu- Via Cintia, 21, Napoli 80126, Italy E-mail: alombard@unina.it tion is introduced to the sample pad, the analyte (in the case E. Renzi, A. Piper, A. Merkoçi of a positive sample) binds to the detection antibody on the Nanobioelectronics & Biosensors Group AuNPs and this conjugate flows through the nitrocellulose in Institut Català de Nanociència I Nanotecnologia (ICN2) the direction of the absorbent pad. When it reaches the TL and CSIC and The Barcelona Institute of Science and Technology (BIST) CL, the AuNP/dAb/analyte complex binds to the capture anti- Campus UAB, Bellaterra, Barcelona 08193, Spain E-mail: arben.merkoci@icn2.cat bodies printed in these lines and the accumulation of AuNPs A. Merkoçi gives rise to a red color. The capture antibodies in the TL will Catalan Institution for Research and Advanced Studies (ICREA) only bind to the AuNP/dAb/analyte complex, whereas those in Pg. Lluís Companys 23, Barcelona 08010, Spain the CL will bind to the AuNP/dAb conjugate, to affirm that the The ORCID identification number(s) for the author(s) of this article solution has wicked the full distance of the strip and that the can be found under https://doi.org/10.1002/smll.202207949. strip has been prepared properly. The intensity of the red color © 2023 The Authors. Small published by Wiley-VCH GmbH. This is an in the TL should be directly proportional to the concentration open access article under the terms of the Creative Commons Attribu- of analyte in the sample solution being tested.[11] With the use tion License, which permits use, distribution and reproduction in any of a proper LFA reader, it is possible to estimate the concentra- medium, provided the original work is properly cited. tion of the analyte in the sample solution and determine the DOI: 10.1002/smll.202207949 limit of detection and sensitivity of the developed assay.[12–14] Small 2023, 2207949 2207949 (1 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com This working principle has been used to develop LFiAs for with anti-human-IgG antibodies and FeMC6*a (Figure 2A). multiple diagnostic applications, including pregnancy tests,[15] The conversion of the TMB chromogen, catalyzed by FeMC6*a COVID-19 antigen tests,[16–18] as well as for safety and environ- (Figure 2B), darkens the test line color thus increasing the LFiA mental analyses.[19,20] sensitivity. To the best of our knowledge, this is the first proof- Despite their many advantages, the LFiAs lack the of-concept example of using an artificial enzyme in LFiAs. The sensitivity required to detect many desirable biomarkers, which benefits and signal enhancement properties obtained when currently have to be detected with laboratory assays.[21,22] Recent substituting natural enzymes with FeMC6*a are discussed. studies have therefore focused on developing signal enhance- Taken together, our results demonstrate that miniaturized ment strategies.[23–26] There are too many of these works to peptide-based artificial enzymes might be exploited in practical discuss in detail here, but some notable methodologies include: applications and open new perspectives in the development enlargement of the particle size by silver coating,[27] coating the of diagnostic devices. Indeed, the possibility of modulating AuNPs with a catalytic metal,[28,29] varying the geometry of the FeMC6*a behaviors by design may offer great opportunities in LFiA components,[30] engineering core-shell multifunctional the optimization of LFiAs. AuNPs,[31] and tuning the flow rate using wax barriers.[32,33] Also, many efforts have been devoted to achieving signal amplification with enzyme-catalyzed chemical reactions,[34] 2. Results and Discussion exploiting the activity of enzymes on AuNP surfaces to con- vert substrates into colored products. For instance, horseradish 2.1. Preparation and Characterization of the peroxidase (HRP) catalyzes the oxidation of colorless 3,3′,5,5′-tetra- AuNPs Decorated with Enzymes and Antibodies methylbenzidine (TMB) to create a blue-colored product. The generation of a color localized at the TL has been shown to The AuNPs were prepared following the Turkevich method,[54] significantly enhance the sensitivity of the assay.[35] by reducing tetrachloroauric acid (HAuCl4) in the presence The suitability of enzymes to catalyze the conversion of chro- of trisodium citrate (Na3-citrate). The citrate-stabilized mogenic substrates into colored products, for the purpose of AuNPs were uniform in size and shape (average diameter of enhancing LFA sensitivity, is well established.[34] However, the 15.9 ± 0.8 nm), and highly monodisperse as ascertained by TEM use of natural enzymes suffers from severe drawbacks, such as analysis (Figure S1, Supporting Information). AuNPs were sub- low stability, loss of activity, and denaturation over long storage sequently functionalized by adsorbing FeMC6*a and the goat times. Thus, many efforts are being devoted to developing anti-human-IgG antibody as dAb (Figure 2A), for analyte detec- artificial enzymes for several applications.[36–40] Among artificial tion. Human-IgG was selected as the target analyte because metalloenzymes, Mimochromes (MCs) were conceived by us as its detection in lateral flow assays has been very well charac- mimics of natural heme-proteins and developed using a minia- terized and developed, both by us[4,35,55] and other groups.[56,57] turization approach followed by iterative re-design steps.[37,41,42] This makes human-IgG an ideal analyte for testing and MCs are composed of two small helical peptide chains cova- validating new sensing technologies, such as that based on the lently linked to the propionic groups of a deuteroporphyrin artificial miniaturized peroxidase FeMC6*a used in this work. IX (DPIX). After several rounds of re-design, shaping both Further, human-IgG detection and quantification are also clini- first- and second-coordination shell interactions[43] and confor- cally relevant, for the diagnosis and progression monitoring of mational constraints,[44] Mimochrome VI*a (MC6*a) was iden- infections.[58] tified as the best scaffold for catalytic purposes. Several interdependent parameters have to be optimized to MC6*a can host several metal ions, affording artificial prepare biomolecule-functionalized AuNPs suitable for incor- enzymes with a tailored catalytic activity.[45–48] In its Fe(III) poration into lateral flow strips.[4] One of these parameters complex, MC6*a acts as a robust and highly active peroxidase is the minimum concentration of the dAb/enzyme mixture (Figure 1A), displaying a 20-fold higher catalytic efficiency in necessary to completely cover the AuNP surface and to stabi- 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) lize the colloid. Furthermore, as biomolecule adsorption onto oxidation than that of wild-type HRP.[44] Despite its simplified AuNPs occurs through electrostatic and/or hydrophobic inter- structure, experimental data have proven the ability of FeMC6*a actions, the pH greatly influences the physisorption as well as to outperform natural and artificial biocatalysts in catalyzing the colloidal stability. Thus, screening for optimal conditions to several oxidation reactions.[49,50] The success of this approach avoid aggregation of the AuNP-biomolecule conjugates is first has paved the way to further widen the functional repertoire of needed. Subsequently, blocking agents should be used to cover FeMC6*a, exploring its catalytic potential after conjugation on the AuNPs surface area not filled with the biomolecules as well gold electrodes and nanomaterials.[51,52] Since FeMC6*a retains as to prevent nonspecific adsorption in the final test. its structure and catalytic behavior when conjugated to gold Therefore, salt-induced gold aggregation tests were performed nanomaterials, the size of this mini-enzyme (radius of gyra- to select the optimal conditions to functionalize AuNPs tion ≈ 1 nm, compared to HRP ≈ 3 nm)[53] grants the possibility with the dAb and FeMC6*a (see Experimental Section and of drastically influencing the specific activity of functionalized Figures S2–S5, Supporting Information). The ratio between nanomaterials, by increasing the active-site density. the dAb and FeMC6*a was set at 9:1 (expressed in µg mL−1), In this work, the immobilization of the artificial peroxidase in order to introduce the catalytic peroxidase functionality FeMC6*a on AuNPs has been exploited as a means of onto AuNPs while keeping a high density of dAb on the gold improving LFiA sensitivity. In particular, we describe the surface for efficient analyte recognition. Bovine serum develop­ment of a two-step LFiA for the detection of human-IgG albumin (BSA) is the most commonly used blocking agent as a model analyte (Figure 1B), using AuNPs doubly decorated in LFAs but was not used in this instance, since the blocking Small 2023, 2207949 2207949 (2 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 1. A) Structure of FeMC6*a obtained using PyMOL software, primary sequences of the tetradeca and deca peptides, and summary of the reactions catalyzed. B) Schematic representation (not to scale) of FeMC6*a–assisted lateral flow two-step immunoassay for human-IgG detection. agent should be smaller than the biomolecules immobilized weight peptide-porphyrin conjugate (radius of gyration ≈ 1 nm, on AuNPs.[4] Considering that FeMC6*a is a low molecular MW ≈ 4 kDa), poly(ethylene glycol) average Mn 400 Da Figure 2. A) Schematic representation of the doubly decorated AuNPs, namely AuNPs@dAb@FeMC6*a, used in the LFiA. B) Changes in the color of a section of the conjugate pad, occurring in the second step of the assay format thanks to FeMC6*a-catalyzed TMB oxidation. Small 2023, 2207949 2207949 (3 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 3. UV–Vis spectra of citrate-AuNPs (black line) and purified, functionalized AuNPs: A) AuNPs@dAb@FeMC6*a, dark cyan line; B) AuNPs@ dAb@HRP, purple line; C) AuNPs@dAb, red line. (PEG-400) was judged to be more suitable since its dimensions compared to natural peroxidases, coupled with the easy scale- are comparable to those of the miniaturized enzyme.[59] Based up of its synthetic route, offers significant advantages for a on the results of the gold aggregation screening, the minimum variety of practical applications.[49,52] To evaluate its potential as dAb/FeMC6*a concentration that gave the highest absorbance a substitute for natural enzymes in LFiA technology, its catalytic difference was 6.5 µg of biomolecules in 1 mL of the AuNPs activity toward the H2O2-mediated TMB oxidation was assayed. stock solution (0.65 µg of FeMC6*a and 5.85 µg of dAb), Indeed, TMB plays an important role as a chromogenic sub- backfilled with a 1% (v/v) aqueous solution of PEG-400 at pH 7 strate in HRP-based immunoassays and has also recently been (Figure S5, Supporting Information). defined as “the chosen substrate” in colorimetric assays, for For comparative purposes, the same conditions were used to its analytical performances as well as molecular properties, functionalize AuNPs with goat anti-human-IgG antibodies and affording high sensitivity and stability.[62] HRP, as well as AuNPs with only goat anti-human-IgG antibodies. Kinetic parameters for the H2O2-dependent TMB oxida- The functionalized AuNPs were then purified by centrifuga- tion catalyzed by FeMC6*a were evaluated and compared to tion and resuspension in the conjugate pad buffer (PBS 10 mM literature data on HRP[63] (see Paragraph S3, Figure S6, and pH 7.4, 5% w/v sucrose, 1% v/v PEG-400, 0.5% v/v Tween-20). Table S1, Supporting Information). FeMC6*a and HRP showed The AuNPs were stable under these conditions, as evidenced similar turnover frequencies (kcat 2600 and 3300 s−1, respectively) by the absorption spectra, which showed no broadening of the toward TMB oxidation. However, because of the increase in surface plasmon resonance band (SPRB), nor any shift in the TMB Km value, FeMC6*a catalytic efficiency (kcat Km−1) showed absorption maximum. Figure 3 shows the UV–Vis spectra of a 4-fold decrease when compared to the natural counterpart. the functionalized AuNPs, namely AuNPs@dAb@FeMC6*a, Nevertheless, this finding was not considered detrimental to AuNPs@dAb@HRP, and AuNPs@dAb. the assay, as it would be counterbalanced by the increase of the For all the systems, a red-shift of 6 nm in the SPRB of the active site density of the immobilized enzyme on AuNPs, thanks citrate-stabilized AuNPs (from 520 to 526 nm) was observed to the reduced size of FeMC6*a with respect to HRP. after functionalization. This finding is consistent with changes One key and critical aspect to employ enzymes in LFiAs is in the refractive index on the surface of the nanoparticles, that the catalysts retain their function after drying and rewet- caused by biomolecule adsorption.[60] ting.[4] Therefore, the stability of the AuNPs@dAb@FeMC6*a TEM images of AuNPs@dAb@FeMC6*a, AuNPs@dAb@ was evaluated by drop casting the solution onto a conjugate HRP and AuNPs@dAb, analyzed after negative staining with the pad, which was dried under vacuum (Figure 2B) and rewet. UranyLess reagent, exhibited a white halo surrounding the gold The drying process did not cause the AuNPs to aggregate core, evidence of the presence of a protein shell (Figure 4).[51,52] (the conjugate pad did not turn a purplish color) and, most After biomolecule conjugation, the size of the AuNP gold core importantly, did not affect the catalytic activity of FeMC6*a. was retained, thus evidencing the colloidal stability of the func- By simply adding TMB solution to the pad, rewet with PBS, tionalized AuNPs. Some clustering, visible in the TEM images, it was possible to observe the change of color from reddish was attributed to the deposition and drying process when to dark purple, thus evidencing the successful oxidation of preparing the samples on the carbon-coated copper grids for the chromogen substrate, catalyzed by the artificial FeMC6*a TEM analysis.[61] This is supported by the UV–Vis spectra of all peroxidase. the conjugates, where no aggregate formation could be observed (Figure 3). The average diameters of the nanoconjugates were measured as 20 ± 2 nm for AuNPs@dAb, 19.8 ± 1.3 nm for 2.3. Performance of the Assay using FeMC6* a and AuNPs@dAb@FeMC6*a, and 26 ± 3 nm for AuNPs@dAb@ Evaluation of the Analytical Sensitivity HRP, indicating that the dAb alone and together with HRP and FeMC6*a are decorating the AuNPs surface (Figure 4). To assess the performance of the artificial FeMC6*a peroxidase in LFiAs, strips were prepared following a protocol established in the literature (see Experimental Section).[4,35] The assay 2.2. Evaluation of FeMC6*a Stability/Activity format consists of two separate human-IgG detection events under Operational Conditions (see Figure 1B). In the first assay step, red bands at the test and control lines of the strips arise from the accumulation of As described above, FeMC6*a is a promiscuous, artificial AuNPs, as is typical in conventional AuNP-based LFiAs. In miniaturized peroxidase.[44,50] The reduced size of this catalyst the second assay step, the TMB substrate was added, which is Small 2023, 2207949 2207949 (4 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 4. TEM images of the AuNPs-biomolecule conjugates, along with the corresponding size distribution histograms: A–C) AuNPs@dAb@ FeMC6*a, D–F) AuNPs@dAb@HRP, and G–I) AuNPs@dAb. The samples were stained with UranyLess solution before observation (N ≈ 150). oxidized by the peroxidase immobilized on AuNPs, an event photographs in Figure 5A,B, even with the naked eye, there is a expected to improve the colorimetric signal intensity. After the clear enhancement of the signal after TMB addition. assembling of the strips with AuNPs@dAb@FeMC6*a, the This is most clearly seen when examining the TL of the first detection was obtained by adding a solution of human-IgG 10 ng mL−1 human-IgG, where a response is undoubtedly seen on the sample pad, allowing the solution to flow, and waiting after the second step (Figure 5B) that was not present after the for 15 min to see the appearance of the colorimetric signal at TL first step (Figure 5A). Quantitative analysis of the strips was and CL. All the experiments were performed in triplicate, for performed by acquiring images of the strips with a scanner, a series of different analyte concentrations (see Experimental and analyzing the strips using “ImageJ” (see Paragraph S4 Section). Images of the lateral flow strips, with different and Figure S7, Supporting Information).[64] From the analysis human-IgG concentrations (between 0 and 1000 ng mL−1), of the strips, test line values were derived for the first and the obtained after the first assay step are provided in Figure 5A. second step of the assay (Figure S8, Supporting Information). As expected, a colored test line appeared in all the positive The derived test line values, background subtracted, were used samples, but not in the blank. The color intensity increased to evaluate the limit of detection (LoD), taking into account: with the concentration of the target, and the lowest analyte i) the values obtained from the blank (LoB), ii) the standard devi- concentration that produced a colored line clearly distin- ation (σ) related to the blank, and iii) the values coming from guishable from the blank by the naked eye was 30 ng mL−1 the lowest concentration of human-IgG (10 ng ml−1), according (Figure 5A). to the following relations: LoB = (mean value)blank + 3σblank; The second step of the assay was carried out by adding 10 µL LoD = LoB + 3σlowest human-IgG concentration.[65] of a TMB-containing solution directly onto the lines. A com- Next, test line values were plotted against the target analyte mercially available, ready-to-use TMB solution was used to concentration, for both the first and second steps of the assay keep the LFiA as simple as possible, avoiding the preparation (Figure 5C,D, respectively). Data fitting with a non-linear of separate solutions of substrate and oxidant. Figure 5B shows Langmuir isotherm (Equation (1) in Experimental Section) photographs of the strips after TMB addition. Comparing the allowed the determination of the LoD as a function of the analyte Small 2023, 2207949 2207949 (5 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 5. LFiA using AuNPs@dAb@FeMC6*a conjugates. Photographs of the strips and assay calibration curves for human-IgG detection acquired A,C) before and B,D) after the addition of the TMB solution, taken with a scanner. All concentrations were repeated in triplicate. concentration. Prior to TMB addition, an LoD of 36.4 ± 1.4 ng mL−1 to the differences in size between the two peroxidases. The was determined (Table 1). Enhancement of the test line inten- reduced size of the artificial peroxidase allows the immobiliza- sity after TMB addition was clearly evidenced by comparing tion of a higher number of active sites on the AuNPs surface. the curves in Figure 5C,D. Data analysis for the second assay This, in turn, enhances the specific activity of AuNPs@dAb@ step, after TMB oxidation on the strips, showed that the LoD FeMC6*a conjugate in TMB oxidation and signal amplification. improved to 8.2 ± 1.2 ng mL−1 (Table 1). To validate this hypothesis, the amount of peroxidase (either FeMC6*a or HRP) in the AuNPs@dAb@FeMC6*a and AuNPs@dAb@HRP conjugates was estimated by quantifying 2.4. Performance of the LFiA: Comparison with a Conventional the heme content, following the method reported by Onoda Assay and when using the HRP-Containing Conjugate et al.[66] In this protocol, the AuNPs are treated with potassium cyanide (KCN), which quenches the SPRB as a consequence of The performances of the LFiAs when using AuNPs@dAb@ converting the Au0 to Au III (CN)−4 , and yields the bis-cyanide FeMC6*a were compared with those based on AuNPs@dAb complex of the heme-containing catalyst, characterized by and AuNPs@dAb@HRP conjugates. The conventional LFiA, an absorption band at 416 nm. Using a known molar extinc- performed with strips prepared using AuNPs@dAb only tion coefficient, it was possible to estimate the enzyme con- (Figure S9, Supporting Information), exhibited a limit of centration from the UV–Vis spectra. Through this procedure detection of 30.5 ± 1.9 ng mL−1 (analysis of the strips is shown (see Experimental Section), the concentration of FeMC6*a in Figure S10, Supporting Information). in the AuNPs@dAb@FeMC6* sample was estimated to be Figure 6 shows photographs of the lateral flow strips 1.6 10−7 M (see Figure S12, Supporting Information). However, prepared with AuNPs@dAb@HRP conjugates, obtained when the identical procedure was performed on the AuNPs@ in a two-step assay format identical to that performed with dAb@HRP conjugate, no absorbance peak was observed, FeMC6*a. As observed for the artificial peroxidase, HRP oxida­ highlighting that the amount of HRP in the sample was not tion of TMB provides an enhancement of the colorimetric spectroscopically detectable, and thus lower than that of signal at the TL (Figure 6A,B). The LoD of the first step was FeMC6*a in the corresponding conjugate sample. found to be 37.4 ± 1.6 and 18.2 ± 0.9 ng mL−1 for the second step Finally, the LoD obtained with the FeMC6*a-based LFiA (after TMB addition), (Figure 6C,D respectively). Analysis of the (8.2 ± 1.2 ng mL−1) is within the range observed for signal- strips is shown in Figure S11, Supporting Information. amplified LFiAs (usually ranging from 0.1 µg mL−1 to 0.1 pg mL−1, The LoD values obtained in all the assay formats are com- as reported by Liu et al.[26]). Further, our assay shows good piled in Table 1. Comparing these values, it can be concluded that the presence of the enzymes, either FeMC6*a or HRP, Table 1. Limit of detection (LoD) values using the different systems on caused a very slight decrease in the performance of the conven- the conjugate pad. LoD values are reported as mean values ± SD (n = 3). tional assay (step 1). This is most probably a result of reducing the total amount of dAb on the nanoparticles, thereby slightly Limit of detection [ng mL−1] decreasing their probability of binding to the target analyte. Label on conjugate release Conventional assay Two-step assay (TMB Interestingly, the lowest LoD value was obtained in the two- pad oxidation) step assay format, exploiting the catalytic activity of FeMC6*a. AuNPs@dAb 30.5 ± 1.9 – This artificial peroxidase gave a higher than four-fold sensitivity AuNPs@dAb@FeMC6*a 36.4 ± 1.4 8.2 ± 1.2 enhancement, whereas a two-fold enhancement was observed AuNPs@dAb@HRP 37.4 ± 1.6 18.2 ± 0.9 with HRP. Such a difference in assay sensitivity may be related Small 2023, 2207949 2207949 (6 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 6. LFiA using AuNPs@dAb@HRP conjugate. Photographs of the strips and assay calibration curves for human-IgG detection acquired A,C) before and B,D) after the addition of the TMB solution, images recorded with a scanner. All concentrations were repeated in triplicate. correlation with other analytical methods to detect human- strips containing both AuNPs@dAb@FeMC6*a and AuNPs@ IgG. LoD values reported for conventional ELISA methods dAb@HRP in the conjugate pad, thus indicating good stability. for IgG quantification range from 1.6 ng mL−1[67] down to 0.24 ng mL−1, as claimed in a commercial kit.[68] More recently, a variety of different platforms for human-IgG detection have 3. Conclusion been reported, such as molecular imprinted SPR chips[69] or optical sensors,[70,71] achieving LoDs of 56,[69] 37,[70] and In this work, we have reported the integration of the synthetic 28 ng mL−1.[71] mini-enzyme FeMC6*a into an LFiA device for the sensitive detection of human-IgG. The enzymatic reaction catalyzed by FeMC6*a exposed on the AuNPs surface allows the oxidation 2.5. Stability Tests of TMB substrate, which in turn enhances the sensitivity of the LFiA with respect to the direct measurement of the AuNPs To evaluate the long-term stability of the lateral-flow strips, tests as non-modified optical labels. All the results reported herein were performed by storing the strips at room temperature prove that FeMC6*a i) tolerates the harsh conditions necessary for 4 weeks. The detection of human-IgG at three different for creating the conjugate pad of a lateral flow strip; ii) does concentrations (0, 50, and 500 ng mL−1) was carried out in not interfere with the binding between human-IgG and the triplicate on days 0, 3, 7, 15, and 30. The strips retained excel- detection/capture antibodies; iii) acts as an efficient catalyst on lent performance for the first 7 days, whereas the optical signal paper, boosting the sensitivity of the enzyme-based LFiA up to decreased by 20% starting from day 15 (Figure S13, Supporting 4-fold with respect to a conventional LFiA. In fact, when using Information). This result was observed right after the first step FeMC6*a as label, the visual detection limits lowered from of the assay, the recognition of human-IgG from the antibody ≈ 40 to ≈ 10 ng mL−1, thus demonstrating an enhancement in molecules. Interestingly, the test line peak value, obtained upon sensitivity. Remarkably, when compared to its natural coun- the addition of TMB solution, showed that FeMC6*a copies terpart (HRP), FeMC6*a grants the assay with a significantly on AuNPs retained their activity over the entire month tested. better limit of detection. Finally, the shelf-life analysis revealed Similar results were obtained by the strips containing HRP- that FeMC6*a functionalized strips are stable for up to a month functionalized AuNPs (Figure S14, Supporting Information). at room temperature. These data suggest that the observed decrease in the optical These encouraging results will inspire future optimization signal is not related to enzyme inactivation, either FeMC6*a or to assay the performance of the FeMC6*a-based LFiAs in real- HRP, but to other factors, for example, loss of dAbs recognition world samples. Currently, the LFiA format has been developed ability over time, probably because of the presence of some sur- and tested in idealized laboratory conditions and buffers to get factants (such as Tween-20), used when preparing lateral flow a fundamental comparison of the performance of FeMC6*a strips to improve the flow across the paper or the release of the with respect to HRP, thus highlighting the advantages of using label particles. Also, the presence of other proteins included on the artificial enzyme. However, complex real-world samples the strips (such as BSA employed as a blocking agent in nitro- (such as blood, saliva, urine, and so on) contain a plethora of cellulose), could have had a detrimental effect on the stability of biomolecules that can non-specifically bind to the lateral flow the strips.[4] Coefficients of variation (CVs) were calculated from assay components and interfere with its performance. Several the ratio of the standard deviation to the average signal. As factors, such as the specificity of the capture and detection reported in Table S2, Supporting Information, the CV values for antibodies, coupled with the correct selection of membrane type concentrations of human-IgG ranged from 5% to 30% for the and any necessary coatings to reduce non-specific adsorption, Small 2023, 2207949 2207949 (7 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com need to be optimized when working on real samples. Such from Imagene Technology (Hanover, Germany). The functionalized optimization is beyond the scope of this work and will be the nanoparticles were purified by centrifugation with an Allegra 64R from Beckman Coulter (Brea, CA, USA). The strips were cut with a lateral subject of future investigations. flow strip cutter from Shanghai Kinbio Tech (Pudong New District, Altogether, these results highlight that the reduced size of Shanghai, China). The UV–Vis spectra were recorded with a SpectraMax FeMC6*a with respect to HRP, coupled with the easy scale- ID3 spectrophotometer from Molecular Devices (San Jose, CA, USA), up of its synthetic route, offers significant advantages for use and with a Cary 60 spectrophotometer (Agilent, CA, USA) using quartz in the construction of bioinspired sensors and nanomaterials. cuvettes with 1.00 cm path lengths. Wavelength scans were performed Traditionally, catalytic methods of signal enhancement in lateral at 25 °C from 200 to 800 nm, with a 600 nm min−1 scan speed. All flow assays have utilized chemical catalysts, enzymes and data were blank corrected. Transmission electron microscopy (TEM) micrographs were acquired in bright field mode using a TEM TECNAI bioengineered enzymes. Moreover, very recently some nano­ G2 20ST (Fei, Hillsboro, OR, USA) operating at 120 kV. Data analysis materials, namely nanozymes,[72,73] have been reported as an was performed with Origin Pro 9.0 software (Origin Lab Corporation, alternative solution. Northampton, MA, USA), and ImageJ software (National Institutes To the best of our knowledge, this is the first study of Health, available free of charge at Web site rsb.info.nih.gov/ij/). All presenting the implementation of a peptide-based artificial the molecular graphics pictures were generated with PyMOL software enzyme to enhance the colorimetric signal in an LFiA. Despite (DeLano Scientific Ltd), and ChemDraw Ultra 12. Materials and Reagents: Polyclonal goat anti-human-IgG antibody significant achievements reached in the field of artificial (I1886), human-IgG whole molecule (12 511), bovine serum albumin metalloenzymes, practical application in catalysis, biotech- (BSA), tetrachloroauric acid solution (HAuCl4, 30% w/w), trisodium nology and sensor technology is still in their infancy. Whilst citrate dihydrate (Na3-citrate), phosphate buffer saline (PBS) tablets, the performance enhancement achieved is not groundbreaking sodium tetraborate, boric acid, sucrose, Tween-20, sodium dodecyl with respect to the obtained LoD, we believe that the introduc- sulfate (SDS), and TMB ready-to-use solution (T0565) were purchased tion of a catalytic signal enhancement, through the use of a from Sigma Aldrich (Madrid, Spain). Chicken anti-goat antibody was purchased from Abcam (ab86245, Cambridge, UK). Polyclonal goat totally synthetic artificial enzyme, is of interest to the field. The anti-human-IgG antibody was used as the detection antibody (dAb) added value of using an artificial enzyme like FeMC6*a, as a and capture antibody on the TL, while chicken anti-goat antibody was valuable alternative with respect to native or mutated enzymes, used as a capture antibody on the CL. Poly(ethylene glycol) average lies in its intrinsic characteristics, and in particular in allowing Mn 400 Da (PEG-400) was purchased from Fluka. 2,2,2-trifluoroethanol to 1) introduce new features and/or improve performance by (TFE) was supplied by Romil (Cambridge, UK). H2O2 solution (50% expanding the range of amino acids beyond those used in v/v) was provided by Merck Life Science. 3,3’,5,5’-tetramethilbenzidine Nature; 2) analyze the effect of mutation on recognition and (TMB) was purchased from Fluka. Peroxidase from horseradish (HRP, P8375-2KU) was purchased as a lyophilized powder from Sigma catalytic activity on smaller and thus easier to study systems; Aldrich (Madrid, Spain). 3) increase the active-site density, because of its smaller dimen- The artificial protein FeMC6*a was synthesized as reported in the sions respect to native enzymes. This last point is crucial literature.[44] especially for applications involving enzyme immobilization, Nitrocellulose membrane (CN150, 1UN15WR100025NT) was as this mini-enzyme allows a drastic increase of the specific purchased from Sartorius Stediem (Göttingen, Germany) and used as activity of functionalized nanomaterials. the detection pad. Cellulose membranes (CFSP001700) were used as sample and absorbent pads and glass fiber (Standard 14, GFCP00900) FeMC6*a has also several advantages with respect to the was used as the conjugate pad. Both were purchased from Millipore nanozymes that mimic HRP activity. As recently highlighted Corporation (Billerica, MA, USA). Adhesive backing cards (MIBA-020) by Ashrafi et al.,[72] even though some nanozymes show inter- were purchased from DCN Dx (Carlsbad, CA, USA). Milli-Q water, esting catalytic behaviors, they suffer from being highly vari- produced using a Milli-Q system (18.2 MΩ cm−1) purchased from able as a result of their means of preparation. Shape, size, and Millipore (Sweden), was used for the preparation of all the solutions. polydispersity may severely change as a consequence of dif- For TEM measurements, copper grids with carbon films (Ted Pella, product 08144-F, 400 mesh) were used and the nanoconjugate samples ferent experimental conditions, thus affecting their catalytic were stained with UranyLess staining solution purchased from Electron behaviors. The artificial miniaturized peroxidase FeMC6*a Microscopy Sciences (Hatfield, PA, USA). holds sufficient molecular behavior to replicate native enzyme Unless otherwise stated, the PBS solution (10 mM, pH 7.4) consisted function, within a defined and predetermined molecular struc- of phosphate buffer(0.01 M), NaCl (0.137 M), and KCl (0.003 M). The ture. Thus, it represents a middle ground between engineered sample pad buffer was made up of PBS containing BSA (5% w/v) and enzymes and nanozymes. Tween-20 (0.05% v/v). The conjugate pad buffer was prepared with PBS, In conclusion, this work is intended as a proof-of-concept sucrose (5% w/v), PEG-400 (1% v/v), and Tween-20 (0.5% v/v). The washing solution contained PBS (5 mM pH 7.4) and SDS (0.01% w/v). and will hopefully inspire the future optimization and devel- Synthesis and Characterization of Citrate-AuNPs: Citrate-capped gold opment of novel Mimochromes which may further enhance nanoparticles were prepared following the Turkevich method.[54] Briefly, the performance of lateral flow assays in real-world samples. an aqueous solution of Na3-citrate (1.25 mL, 1% w/v) was added to a This will allow moving away toward assay formats where the boiling aqueous solution of HAuCl4 (50 mL, 0.25 mM) under vigorous response can be enhanced by the intelligent design of artificial stirring. The addition of the reducing agent induced the formation of enzymes. gold nanoparticles, evidenced by a color change from light yellow to bright red. After that, the solution was allowed to boil for an additional 10 min, under stirring, and finally to cool down to room temperature. The as-synthesized colloidal solution of citrate-AuNPs was stored at 4 °C 4. Experimental Section in the dark until further use. The concentration of the citrate-AuNPs in solution was qualitatively evaluated by UV–Vis spectroscopy (Figure S1A, Instruments: The test line and the control line were dispended on Supporting Information) following a method reported by Fernig et al.[74] the nitrocellulose membrane using an IsoFlow Bioreagent dispenser The ratio between the maximum absorbance of the SPRB (ASPRB) and the Small 2023, 2207949 2207949 (8 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com maximum absorbance at 450 nm (A450) provides a qualitative indication 2 min. After that, the excess solution was removed with filter paper, and of the AuNPs diameter, and hence of its molar extinction coefficient. For the grid was rinsed with Milli-Q water. The grids were allowed to air-dry an ASPRB/A450 ratio of 1.66, the diameter of the AuNPs was estimated to at room temperature overnight.[78] be ≈ 16 nm. From the value of A450 and using the calculated extinction Fabrication of the Strips: The printing of the test and control lines on coefficient (ε450) of 2.67 × 108 M−1 cm−1, the citrate-stabilized AuNP the nitrocellulose membrane (CN150) was performed with a bioreagent concentration in the stock solution was estimated to be 1.67 nM. dispenser using goat anti-human-IgG (1 mg mL−1 in phosphate buffer Salt-Induced Gold Aggregation Tests: During this screening, several 10 mM, pH 7.4) and chicken to goat antibody (1 mg mL−1 in phosphate parameters were taken into account: i) the pH of the AuNP solution, buffer 10 mM, pH 7.4) respectively. The lines were then dried in the oven ii) the concentration and composition of the biomolecule mixture at 37 °C for 2 h. After drying, the nitrocellulose membrane was blocked (containing goat anti-human-IgG antibody as dAb and FeMC6*a), iii) the with a solution of BSA (2% w/v) in PBS for 20 min and washed two addition of a blocking agent, and iv) the concentration of the blocking times using the washing solution (15 min, two times). The conjugate agent. The pH of citrate-AuNPs solution was adjusted until 7, 8, and pad was prepared by dispensing the conjugate solution, containing 9 using borate buffer (100 mM, pH 9.2). Then, aliquots of citrate-AuNPs either AuNPs@dAb@FeMC6*a, AuNPs@dAb@HRP or AuNPs@dAb, at the chosen pHs (150 µL) were incubated with aqueous solutions of depending on the selected format assays, on the glass fiber and drying dAb and FeMC6*a at several concentrations (10 µL, concentrations: 200, it in a vacuum chamber at room temperature for 2 h. The sample pads 175, 150, 125, 100, 80, 60, 40, and 0 µg mL−1, of which 10% was FeMC6*a were pre-treated by soaking the cellulose membrane in the sample pad and 90% was dAb). After that, the blocking process was performed buffer and dried in the oven at 37 °C for 3 h. Finally, the assembling using an aqueous solution of PEG-400 (10 µL; 0, 1, 2, and 3% v/v) and of the pads was guided by laminated cards, already containing the the test ended with the addition of a solution of NaCl (20 µL, 10% w/v). nitrocellulose membranes, and cut into 0.3 cm-width strips with a lateral Each step was carried out for 20 min at 25 °C, 650 rpm. Finally, flow cutter. The size of the strips was fixed at 6 × 0.3 cm, with a bed UV–Vis spectra were recorded, and the degree of aggregation was volume of 90 µL. measured by the red-shift of the SPR bands relative to the 520 nm value Procedure of the Assay: The assay was conceived to be performed obtained from the stable solution of citrate-AuNPs (Figures S2–S4, in two steps: i) the appearance of the CL and TL because of the flow Supporting Information). The difference between the absorbance at of human-IgG solutions on the LFiA strips; ii) the addition of TMB 520 nm and at 580 nm (taken as the absorbance value for aggregated solution on the colored lines. For the first step, solutions of human-IgG AuNPs) was evaluated and plotted against the concentration used (90 µL, concentrations 0, 10, 30, 50, 75, 100, 300, 500, 750, and 1000 ng (Figure S5A–C, Supporting Information).[75] mL−1, prepared in PBS) were dispensed on the sample pad. PBS without Functionalization of AuNPs: The physisorption of goat anti-human-IgG an analyte was used as a blank. All the measurements were carried out antibody (dAb) and FeMC6*a on AuNPs was performed as follows, using in triplicate. the optimal conditions elucidated by the gold aggregation tests and The solution was allowed to reach the absorbent pad and the strips referring to some procedures reported in the literature.[76,77] Prior to this were left under ambient conditions for 15 min to allow the lines to procedure, separate stock solutions of dAb and FeMC6*a (2.0 mg mL−1) develop. Then, the strips were washed with PBS (70 µL) to improve the were prepared in Milli-Q water. Then, a solution containing both the background color. For the second step, the ready-to-use solution of the biomolecules (600 µL, 110 µg mL−1 in Milli-Q water containing 6.6 µg chromogen TMB was added directly to the test line and the control line of FeMC6*a and 59.4 µg of dAb) was added to an AuNP solution (10 µL) and a period of time of 10 min was waited to allow the oxidative (9.0 mL, pH 7 adjusted with borate buffer 100 mM, pH 9.2) at 650 rpm conversion of the chromogenic substrate. An enhancement of the red and 25 °C for 90 min. To this, a solution of PEG-400 (600 µL, 1% v/v color of the lines was ascertained by the naked eye. After each step, in Milli-Q water) was added and incubated under the same conditions images of the strips were acquired using a SkanMulti LFA scanner and for 30 min. The sample of functionalized AuNPs was then purified the quantitative data analysis was performed with ImageJ software, in by centrifugation (10 000 rpm, 40 min, 4 °C). After that, the clear order to construct calibration curves. Details about the analysis of the supernatant was discarded and the collected pellet was re-suspended in strips are given in the Supporting Information (Figures S7, S8, S10, and 3.0 mL of the conjugate pad buffer, to afford AuNPs@dAb@FeMC6*a. S11, Supporting Information). The same synthetic and purification procedures were performed using Each set of data points was fitted to the non-linear Langmuir HRP (stock solution 2.0 mg mL−1 in Milli-Q water) instead of FeMC6*a, isotherm equation. This model initially devised to describe the to give AuNPs@dAb@HRP. adsorption behavior of gaseous molecules to a solid substrate as a Likewise, the AuNPs@dAb sample was prepared by incubating function of gas pressure, is now widely exploited for the quantification of the AuNPs solution (9.0 mL) with the goat anti-human-IgG antibody the number of molecular adsorbates on a solid substrate as a function solution (600 µL, 110 µg mL−1 in Milli-Q water) and incubated at of the concentration of the molecule in solution.[79,80] In this case, the 650 rpm and 25 °C for 30 min. Next, a solution of PEG-400 (600 µL, solid-liquid interaction between the capture antibody on the test line and 1% v /v in Milli-Q water) was added to the mixture and incubated at the antigen bound to the detection antibody (carried along the strip by the same conditions. After that, the excess reagents were removed by AuNPs) could be modeled. From previous studies, it was known that centrifugation (10 000 rpm, 30 min, 4 °C) and the collected precipitate there was a 1:1 stoichiometric binding ratio between the antibody and was dispersed in 3.0 mL of the conjugate pad buffer. the human-IgG. The following equation was used: After purification, UV–Vis spectra were acquired by diluting the sample three times with the conjugate pad buffer (Figure 3). No θ mK [Human − IgG ] θ= (1) broadening of the bands or loss of nanomaterial was observed, as the 1+ K [Human − IgG ] absorbance values recorded are the same as those of citrate-AuNPs, indicating that the concentration of AuNPs in the undiluted stock where θ is the fraction of human-IgG molecules bound by goat anti- solutions is ≈ 5 nM for AuNPs@dAb@FeMC6*a, AuNPs@dAb@HRP, human-IgG antibody (the capture antibody on the test line); θm is the and AuNPs@dAb. maximum number of bound human-IgG molecules; [Human-IgG] is the Characterization of Functionalized AuNP Samples: Transmission concentration of analyte in ng mL−1, and K is the Langmuir constant, electron microscopy (TEM) micrographs were acquired in bright field which is a parameter indicating the strength of the interaction between mode. The samples for TEM analyses were prepared by loading the the antibody and the antigen. θ is related to the test line optical value functionalized AuNP solutions (5 µL) onto carbon-coated copper grids evaluated with ImageJ (background subtracted) and θm is the test line for 10 min and removing the excess solution with filter paper. High optical value related to the highest concentration of human-IgG. negative contrast, to visualize the biomolecular shell around AuNPs, was Evaluation of the Iron Content with Cyanide Method: Iron content in afforded with UranyLess staining solution. The staining solution (5 µL) AuNPs@dAb@FeMC6*a and AuNPs@dAb@HRP conjugate samples was deposited on the grid and the staining procedure was performed for was evaluated as reported in the literature.[66] The procedure was based Small 2023, 2207949 2207949 (9 of 11) © 2023 The Authors. Small published by Wiley-VCH GmbH 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202207949 by Readcube (Labtiva Inc.), Wiley Online Library on [27/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com on the disruption of the gold core and the consequent formation of a Data Availability Statement bis-cyanide-heme complex of the heme-containing peroxidases (FeMC6*a and HRP). The absorbance of this complex was directly The data that support the findings of this study are available from the proportional to the concentration of heme, thus of the enzyme in the corresponding author upon reasonable request. conjugate sample. The molar extinction coefficient for the bis-cyanide- heme complex formed with FeMC6*a was first evaluated by performing cyanide binding experiments. In detail, a stock solution of FeMC6*a (3.5 µL, 1.1 × 10−3 M in H2O 0.1% v/v TFA) was mixed with a stock Keywords aqueous solution of KCN (196.5 µL, 2.5 × 10−5 or 2.5 × 10−2 M, pH 12). artificial peroxidase, gold nanoparticles, lateral flow immunoassays, The addition of a 1.25-fold molar excess of cyanide ions led to the mono- nanomaterials, sensitivity cyanide adduct with the ferric heme of the artificial enzyme, which Soret band was positioned at 406 nm (Figure S12A, Supporting Information, Received: December 19, 2022 blue line).[81] On the contrary, the addition of a 125-fold molar excess Revised: February 21, 2023 of KCN gave rise to the bis-cyanide-heme adduct, with a Soret band Published online: at 416 nm and a single Q band at 550 nm (Figure S12A, Supporting Information, purple line), as similar UV–Vis response was observed for the bis-cyanide complex of natural heme-proteins.[82] The absorption of the bis-cyanide-heme adduct was employed to evaluate the value of the molar extinction coefficient at 416 nm (ε416 = 61 754 M−1 cm−1). Regarding [1] C. Parolo, A. Merkoçi, Chem. Soc. Rev. 2013, 42, 450. the experiment with the conjugate samples, aliquots of AuNPs@dAb@ [2] S. Sharma, J. Zapatero-Rodríguez, P. Estrela, R. O’Kennedy, FeMC6*a and AuNPs@dAb@HRP (750 µL each) were concentrated Biosensors 2015, 5, 577. by centrifugation (10 000 rpm, 30 min, 4 °C) and the collected [3] K. M. Koczula, A. Gallotta, Essays Biochem. 2016, 60, 111. precipitate (20 µL) was treated with an aqueous solution of KCN [4] C. Parolo, A. Sena-Torralba, J. F. Bergua, E. Calucho, (80 µL, 2.5 × 10−2 M). After an incubation time of 20 min, to allow C. Fuentes-Chust, L. Hu, L. Rivas, R. Álvarez-Diduk, E. P. Nguyen, the disintegration of the gold core, UV–Vis spectra were recorded and S. Cinti, D. Quesada-González, A. Merkoçi, Nat. Protoc. 2020, 15, the absorption at 416 nm was used to evaluate the iron content in the 3788. conjugates. The experiments were repeated in triplicate. [5] D. Quesada-González, A. Merkoçi, Biosens. Bioelectron. 2015, 73, 47. Stability Assay: The stability assay was carried out at room [6] A. Sena-Torralba, R. Álvarez-Diduk, C. Parolo, A. Piper, A. Merkoçi, temperature. The strips were stored under Ar, in sealed bags in the Chem. Rev. 2022, 122, 14881. dark. The detection of human-IgG, before and after the addition [7] B. Khlebtsov, N. Khlebtsov, Nanotechnology 2008, 19, 435703. of TMB solution, was performed on days 0, 3, 5, 7, and 30 using [8] W. Ren, D. R. Ballou, R. FitzGerald, J. Irudayaraj, Biosens. Bioelectron. solutions of analyte at three different concentrations (0, 50, and 2019, 126, 324. 500 ng mL−1 in PBS). The images of the strips were taken with a SkanMulti LFA scanner and the optical intensity of the test line was [9] F. Di Nardo, M. Chiarello, S. Cavalera, C. Baggiani, L. Anfossi, evaluated with ImageJ software. The results of the stability assays are Sensors 2021, 21, 5185. given in the supporting information (Figures S13 and S14, Supporting [10] A.-C. 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