Ultrasensitive detection of lysozyme in droplet-based

Biosensors and Bioelectronics 104 (2018) 8–14

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Ultrasensitive detection of lysozyme in droplet-based microfluidic devices a

b

Maria Chiara Giuffrida , Giovanni Cigliana , Giuseppe Spoto

a,c,⁎

T

Consorzio Interuniversitario “Istituto Nazionale Biostrutture e Biosistemi”, c/o Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, Catania, Italy Clinical Pathology Unit, Regina Elena National Cancer Institute, Via Chianesi, Roma, Italy c Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, I-95125 Catania, Italy a

b

A R T I C L E I N F O

A B S T R A C T

Keywords: Aptamer Chemiluminescence Droplet microfluidics Gold nanoparticles Lysozyme

Lysozyme (LYS) is a bacteriolytic enzyme, available in secretions such as saliva, tears and human milk. LYS is an important defence molecule of the innate immune system, and its overexpression can be a consequence of diseases such as leukemia, kidney disease and sarcoidosis. This paper reports on a digital microfluidic-based approach that combines the gold nanoparticle-enhanced chemiluminescence with aptamer interaction to detect human lysozyme into droplets 20 nanoliters in volume. The described method allows identifying LYS with a 44.6 femtomolar limit of detection, using sample volume as low as 1 μL and detection time in the range of 10 min. We used luminol to generate the chemiluminescence and demonstrated that the compartmentalization of LYS in droplets also comprising gold nanoparticles provided enhanced luminescence. We functionalized the gold nanoparticles with a thiolated aptamer to achieve the required selectivity that allowed us to detect LYS in human serum.

1. Introduction

2017). Lysozyme (LYS) is a relatively small bacteriolytic enzyme playing an essential role in protecting organisms against microbial invasion (Durek et al., 2007; Ercan and Demirci, 2016). It is composed of 129 amino acids and is folded to form a globular structure with an active site where the intramolecular interactions involving Glu35 and Asp52 residues play a central role (Held and vanSmaalen, 2014). The detection of lysozyme circulating in body fluids has significant implications in clinical diagnostics (Guder and Hofmann, 2008). In fact, the over-expression of LYS in serum and urine can be a consequence of diseases such as leukemia (Levinson et al., 2002), chronic kidney disease (Glorieux et al., 2015) and sarcoidosis (Li et al., 2014). More recently, Sawaya et al. described the role played by LYS in the formation of amyloid fibrils (Sawaya et al., 2007). In addition, LYS is a valuable model system for the development of innovative approaches for protein detection. Enzyme-linked immunosorbent assay (ELISA) is the most widely used method to detect human LYS with an assay time of about 4 h, LOD of about 9 pM and sample volume in the 50 μL − 100 μL range (〈http://www.abcam.com/human-lysozyme-elisa-kit-lzm-ab108880references.html〉). Different approaches have been investigated to achieve better performances in LYS detection (Sener et al., 2010; Chen et al., 2008; Jing et al., 2011; Schneider et al., 2010; Vasilescu et al., 2016). In this perspective, chemiluminescence (CL) has been proven an

Droplet microfluidics has emerged as an innovative technology for the development of new devices for medical research, therapeutics and diagnostics (Mashaghi et al., 2016; Bellassai and Spoto, 2016; Giuffrida and Spoto, 2017; Mazutis et al., 2013). It exploits discrete fractions of water solutions isolated from walls of microfluidic channels by a waterimmiscible fluid to obtain nanoliter−femtoliter droplets acting as individual chemical reactors. The compartmentalization of reacting species in droplets offers substantial advantages in high-throughput detection. The compartmentalization also reduces the importance of issues related to the sample consumption and contamination, by allowing the control of the concentration of reacting species and the mixing time of reagents. The implementation of genetic assays on droplet microfluidic platforms has produced significant improvements in nucleic acid-based diagnostics (Giuffrida and Spoto, 2017; Duncombe et al., 2015; Giuffrida et al., 2015). The development of efficient assays for the detection of proteins from droplets instead requires additional investigations to meet requirements of the most demanding applications (Duncombe et al., 2015; Li et al., 2015; Guo et al., 2012). In this perspective, the exploitation of optical and electronic properties of nanostructured materials provides new opportunities for signal enhancement in biosensing (Cao et al., 2011; Lei and Ju, 2012; D'Agata et al.,

⁎

Corresponding author at: Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, Catania, Italy. E-mail address: [email protected] (G. Spoto).

https://doi.org/10.1016/j.bios.2017.12.042 Received 31 July 2017; Received in revised form 20 December 2017; Accepted 26 December 2017 Available online 26 December 2017 0956-5663/ © 2017 Elsevier B.V. All rights reserved.

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Assay sensitivity in the fM − nM range is essential to detect lysozyme available in body fluids or food matrices (Vasilescu et al., 2016). Lysozyme is available at different concentrations in different body fluids, while lysozyme present in food matrices even in trace amount can be responsible for allergic reactions in sensitive individuals. Lysozyme is used in alcoholic fermentation for its ability to control the growth of Gram-positive and spoilage bacteria, without inhibiting yeast growth, and it allows a reduction in the use of sulphur dioxide. In addition, lysozyme is often used as fining agent to remove excess tannins. Lysozyme is classified as a hidden allergen and has been found in different food matrices at the low pM concentration.

efficient approach thanks to the short assay time, the simple instrumentation required and the high sensitivity achievable (Hun et al., 2010; Li et al., 2011). In particular, the interaction of luminol (3-aminophthalhydrazide) with LYS has been demonstrated to enhance luminol CL intensity (Wang and Song, 2010), thus providing a useful luminescence probe for LYS detection. Gold nanoparticles (AuNPs) are widely used for signal enhancement purposes in biosensing (Lei and Ju, 2012; Bertucci et al., 2015; Zanoli et al., 2012). In particular, AuNPs are helpful to enhance luminol CL intensity. AuNPs catalyse the decomposition of the H2O2 oxidising agent to produce hydroxyl radicals which react with luminol thus facilitating the formation of luminol radicals (Zhang et al., 2005). Karabchevsky et al. have recently proposed two additional mechanisms for the luminol chemiluminescence enhancement by AuNPs: i) a plasmonic-mediated antenna effect established when luminol molecules are in close proximity to AuNPs surface and ii) a far-field scattering of photons emitted by luminol (Karabchevsky et al., 2016). In this paper, we show that the integration of AuNPs-enhanced CL detection in droplet microfluidic devices, combined with the aptamerdriven specific adsorption of LYS on AuNPs surface, can be used to detect LYS with a limit of detection (LOD) of 44.6 fM, sample volumes as low as 1 μL and assay time in the 10 − 20 min range. Here, we also show results obtained using the droplet microfluidic-based method to detect LYS in serum obtained from a healthy donor and chronic lymphocytic leukemia patients. Cox and Ellington identified anti-Lysozyme aptamers in 2001 (KD = 31 nM) (Colin Cox and Ellington, 2001; Kirby et al., 2004). Since then, aptamer probes have been used to design biosensing approaches for LYS detection operating with detection limits in the 0.5 nM (Subramanian et al., 2013) − 862 nM (Rohrbach et al., 2012) range. Electrochemical aptasensors for lysozyme detection have been widely investigated and methods able to operate with LOD ranging from fM to nM have been described (Vasilescu et al., 2016). More recently, the anti-lysozyme aptamer has been combined with a rolling circle amplification step and CdTe/CdSe quantum dot fluorescence detection to achieve a 2.6 nM detection limit (Qiu et al., 2017). Traditional biosensing approaches suffers from limitations arising from the non-specific interaction of material dispersed in the biological fluid with the biosensor solid surface. A novel lysozyme sensing concept based on the surface plasmon resonance detection and using Micrococcus lysodeikticus whole cells has been recently demonstrated to be able to detect lysozyme in serum sample with 3.5 nM LOD (Vasilescu et al. (2017). In this case, the integrity of the bacterial cell wall is affected by the exposure to lysozyme available in the serum sample causing the desorption of the whole cell from the sensor surface. Here, we demonstrate that the exploitation of the specific recognition properties of aptamers combined with the AuNPs capacity to enhance CL and compartmentalization in droplets provides superior performances in LYS detection. In addition, limitations of the biosensing detection caused by the non-specific adsorption of components of biological fluids on the biosensor solid surface are avoided when operating in the droplet-confined fluid environment here described. The ability to perform clinical diagnostic assays on small scales using droplet microfluidic devices provides numerous benefits, including reduced quantities of reagents and waste as well as increased controllability of assays (Mashaghi et al., 2016; Giuffrida and Spoto, 2017). Similar benefits are of great importance when the new assay helps in eliminating drawbacks of traditional methods. Typical disadvantages of techniques used in protein detection are linked to the limited throughput and low sensitivity of some of the conventional methods. In addition, the sample contamination is often a serious issue. ELISA is currently the most popular diagnostic tool used for the detection of lysozyme present both in human fluids as well as food matrices. The replacement of the ELISA assay with a droplet microfluidicbased assay can help in further improving the throughput of the analysis and in reducing the potential influence sample contaminations.

2. Experimental 2.1. Materials and reagents We purchased Human lysozyme (H-LYS), lysozyme from chicken egg white (C-LYS) and 5’-Thiol modifier anti-lysozyme aptamer (5’ATCTACGAATTCATCAGGGCTAAAGAGTGCAGAGTTACTTAG-3’) from Thermo Fisher Scientific (Italy). Luminol, potassium periodate (KIO4), potassium ferrocyanide (K4Fe(CN)6), trisodium citrate dihydratetetrachloroauric(III) acid (HAuCl4 3H2O), 1 H,1 H,2 H, 2H-perfluoro-1-octanol (PFO) 97%,tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), tris acetate salt (tris (hydroxymethyl) aminomethane acetate salt), albumin from human serum (HSA), and NaCl, were purchased from Sigma-Aldrich (Italy). 3 M™ Fluorinert™ Electronic Liquid FC3283 was obtained from 3 M (Italy). We performed all experiments using ultrapure water (Milli-Q Element, Millipore. Italy). 2.2. Fabrication of microfluidic devices and droplet generation Microfluidic devices were fabricated in polydimethylsiloxane (PDMS) by replica moulding as described elsewhere (Grasso et al., 2009). The mould was then irreversibly bonded to a microscope cover glass by air plasma etching (Femto low-pressure plasma system.40 kHz generator. Diener electronic GmbH + Co. KG. Germany). The assembled device was maintained at 60 °C for 30 min and used after 24 h to allow the recovery of the hydrophobic surface necessary for the production of water-in-oil droplets. We used two Harvard 33 Twin syringe pumps (Harvard Apparatus. U.S.A.) handling syringes (Hamilton microsyringes: model 1750 - volume 500 μL- and model 1725 -volume 250 μL) connected by Pharmed BPT tubing (ID = 0.25 mm, Cole-Parmer) to the microfluidic device to control the liquid flow rate. We used Hamilton microliter syringes (model 7101. Maximum volume 1 μL. Hamilton. U.S.A.) to inject sample solutions (1 μL) and functionalized AuNPs dispersions (1 μL) into the microfluidic device. Fig. 1 shows a schematics of the microfluidic devices used to perform the experiments here described. Fig. 1 also indicates flow rates of the carrier (FC-3283/PFO 10:1 mixture) and the water fraction used during our tests. We performed the experiments at room temperature. We used a Leica DM IL Fluo inverted microscope equipped with a Leica DFC 450C digital camera and a Lumen 200 (Prior Scientific Inc.) metal-halide lamp to detect luminescence generated from droplets. Image J 1.42 software was used to analyse optical images and to quantify the intensity of the detected luminescence. We defined a region of interest (ROI) inside each droplet, to obtain the average intensity of luminescence emitted from each droplet as pixel values. We calculated the referenced luminescence intensity by subtracting the luminescence detected from droplets comprising only the blank solution from the luminescence generated in the presence of the target molecules. We averaged each intensity value over eight different droplets. We designed our experiments to target human LYS (H-LYS) and performed control experiments using HSA and LYS from chicken egg white (C-LYS). C-LYS has about 60% of similarity with H-LYS sequence 9

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Fig. 1. a) Schematic drawing of a microfluidic device. Experimental conditions used for the experiments are also shown. b) Optical image of droplets.

= −61.3 ± 0.7 mV. A number of aptamer units per AuNPs at the 102 level can be estimated on the basis of data reported for similar systems (Demers et al., 2000). Fig. 2 displays a schematic representation of the binding of lysozyme by apt-AuNPs.

(BLASTP 2.2.31+). 2.3. Synthesis and functionalization of AuNPs We synthesised AuNPs according to the citrate reduction method elsewhere described (Turkevich et al., 1951) and characterised AuNPs dispersions using optical absorption spectroscopy (Nanodrop™ 1000. Thermo Scientific), zeta potential (ζ), dynamic light scattering (DLS) (Zetasizer Nano ZS ZEN3600. Malvern Instruments, Malvern, UK). AuNPs dispersions provided λmax = 520 nm and ζ = −43.4 ± 0.8 mV. AuNPs mean physical diameter was 14.1 ± 0.4 nm (D'Agata et al., 2017). We functionalized AuNPs with 5’-thiol modified aptamer (apt. 5’ATCTACGAATTCATCAGGGCTAAAGAGTGCAGAGTTACTTAG-3’; 5’: Thio C6 S-S: O-(Dimethoxytrityloxy-hexyl-dithiohexyl)-O′-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite) (Hun et al., 2010; Haiping et al., 2009) according to procedures elsewhere described (Liu and Lu, 2006). Briefly, 1 μL of 500 mmol L−1 acetate buffer (pH 5.2) and 1.5 μL of 10 mmol L−1 tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) were added to a tube containing 9 μL of a 1 mmol L−1 anti-lysozyme aptamer solution to activate the thiol-modified DNA strand of the aptamer. We incubated the mixture at room temperature for one hour. Then, we added 3 ml of the previously prepared AuNPs dispersion and put the sample under stirring for at least 16 h at room temperature. We added tris-acetate buffer (pH 8.2, final concentration 5 mmol L−1) and NaCl (1 mol L−1) to the dispersion of the aptamer-modified AuNPs and centrifuged the tube after one day (16.110g at room temperature). The supernatant was pipetted off to remove the free aptamer still dissolved in solution. We dispersed the apt modified AuNPs (apt-AuNPs) in a tris acetate buffer (25 mmol L−1, pH 8.2) containing NaCl (100 mmol L−1). apt-AuNPs dispersions were characterised by λmax = 535 nm and ζ

2.4. Biological samples We obtained human serum from blood samples of selected in-patients of the "Regina Elena National Cancer Institute" (Rome, Italy) during their defined clinical iter. We conducted our study according to the guidelines of the Declaration of Helsinki (1964). The collected blood samples were centrifuged at 2000g for 10 min and serum divided in aliquots before being frozen at −80 °C and stored until analysis. Samples were thawed only once, keeping them at room temperature and immediately analysed. The analysis was performed by operators without knowledge of the clinical history of the samples. We selected serum samples by considering the total white blood cell count and classified samples as ‘normal’ and ‘pathological’. We diluted each serum sample with water (1:100) before the analysis. 3. Results and discussion We obtained a stable generation of droplets at the T-junction of the microfluidic device (Fig. 1) by injecting a mixture of FC-3283 and PFO (10:1), acting as the carrier (flow rate 0.5 μL min−1), and aqueous solutions (flow rate 0.02 μL min−1). Under such experimental conditions, we produced with a frequency of about 0.2 Hz droplets which were about 20 nL in volume. We initially designed the microfluidic device to introduce a time gap of 25 min between the generation of droplets and the detection of the luminescence emitted from them. Fig. 2. Schematic representation of the binding of lysozyme by apt-AuNPs.

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Referenced CL Intensity (a.u.)

The capillary number (Ca) characterises droplet formation in a multiphase flow. Ca is defined as Ca = ην/γ, where η (kg m−1 s−1) is the viscosity, ν (m s−1) is the velocity and γ (kg s−2) is the surface tension between the aqueous phase and the immiscible carrier. We operated under experimental conditions defining low Ca values (Ca = 3.9×10−4) to obtain the rapid separation of droplets from the Tjunction, a stable and reproducible droplet generation frequency and the separation between adjacent droplets (Tice et al., 2004). 3.1. Detection of human lysozyme

Referenced CL intensity (a.u.)

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150

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CL enhancement factor

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4.0 3.5 3.0 2.5 2.0

H-LYS+AuNPS/H-LYS

100

150

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Fig. 4. a) Average relative CL intensity measured from droplets with H-LYS and AuNPs (spheres) and droplets with C-LYS and AuNPs (triangles). LYS concentrations in the 20 fM − 300 fM range produced discriminated signals (two-tailed t-test, level 95%, ν = 8, n = 5 p < 0.0001). Error bars represent the confidence interval of the mean at the 95% level. b) CL enhancement factor as a function of H-LYS concentration.

originating from a selected sample showed a low-variance distribution (%CV < 1). The detected CL intensity was higher for droplets comprising both H-LYS and AuNPs (Fig. 4a) compared with droplets with no AuNPs (Fig. 3) with an enhancement factor (EF) that was linked to the concentration of H-LYS with a saturation dependence (Fig. 4b). We performed additional experiments to test the specificity of the assay. With this aim, we compared CL produced in the presence of both AuNPs and H-LYS (Fig. 4a. Spheres), with CL measured replacing H-LYS with the lysozyme from chicken egg white (C-LYS) used as the control system (Fig. 4a. Triangles). The overlap of CL measured by analysing HLYS and C-LYS demonstrated the lack of specificity of the assay. 3.3. Selective detection of human lysozyme by aptamer-modified AuNPs Fig. 5a illustrates representative results we obtained by modifying the detection process to reduce the delay between the droplet formation and CL detection down to 10 min. We achieved the desired specificity by functionalizing AuNPs with the anti-lysozyme aptamer (apt-AuNPs). Fig. 5a shows the average referenced CL detected from droplets carrying H-LYS (black spheres), CLYS (black triangles) or HSA (grey spheres) in the presence of aptAuNPs. Concentrations investigated were in this case in the 10 fM − 300 fM range. We selected the two control systems to evaluate responses generated by both the most abundant protein in the human blood (human serum albumin, HSA) as well as a protein (C-LYS) having close similarity to H-LYS. The CL intensity detected when H-LYS interacted with apt-AuNPs was similar to the signal detected using not functionalized AuNPs under the same experimental conditions (compare Fig. 5a with Fig. 4a, black spheres). Instead, we observed a significantly different CL intensity when detecting C-LYS or HSA control systems (compare Fig. 5a with Fig. 4a). Even the lowest concentration we investigated (10 fM)

18 16 14

250

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Conc. (fM)

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H-LYS + AuNPs C-LYS + AuNPs

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22

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1.5

H-LYS

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60

4.5

We used AuNPs to enhance the intensity of CL generated from droplets. In particular, we modified the experimental conditions by adding AuNPs (1 μL, 11 nM) to the components described below (Fig. 1. Inlet 5). Fig. 4a shows the average values of the referenced CL we detected from droplets comprising differently concentrated H-LYS solutions (black spheres. Error bars represent the confidence interval of the mean at the 95% level. Two-tailed t-test, level 95%, ν = 8, p < 0.0001). We referenced the detected CL to the luminescence detected from droplets comprising both the blank solution and AuNPs. The concentration of AuNPs in the reference droplets was the same than in droplets carrying lysozyme samples. CL detected from droplets

50

80

Conc. (fM)

3.2. AuNPs enhancement of chemiluminescence

12

a)

50

We performed preliminary experiments aimed at investigating the possibility of detecting CL generated in the microfluidic device after the interaction between LYS and luminol under static conditions. In particular, we compared the luminescence detected from microchannels filled with the blank solution (luminol 0.25 mol L−1 in NaOH 0.5 mol L−1, KIO4 20 mmol L−1 and K4[Fe(CN)6] 3.6·10−4 mol L−1) with that generated from a solution obtained by adding H-LYS to the blank solution. A 2.5 μM H-LYS solution produced a 14.5-fold higher CL signal level compared to the blank solution. The positive outcome of the preliminary experiments allowed us to implement the H-LYS detection in the droplet microfluidics device. We obtained optimal conditions for H-LYS detection by introducing luminol (30 mmol L−1 in NaOH 0.5 mol L−1) from inlet 1, the oxidative and catalytic agents (a 1:5 mixture of K4[Fe(CN)6] 2.3 mmol L−1 and H2O2 3%) from inlet 2, H2O from inlet 3, and 1 μL of H-LYS water solution from inlet 4, respectively (Fig. 1). Fig. 3 shows results obtained by analysing H–LYS solutions in the 20 fM − 300 fM concentration range. In particular, we got data illustrated in Fig. 3 by referring CL detected from droplets with H-LYS to CL generated by droplets carrying the blank solution. Fig. 3 shows the dependence of the detected signals from the H-LYS concentration.

24

100

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Conc. (fM) Fig. 3. Average referenced CL intensity detected from droplets of H-LYS solutions. The detected CL signals allow to discriminate among the differently concentrated H-LYS solutions (two-tailed t-test, level 95%, ν = 8, n = 5, p < 0.0001). Error bars represent the confidence interval of the mean at the 95% level.

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Referenced CL intensity (a.u.)

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100

while analysing H-LYS and apt-AuNPs (5 samples). We referenced CL to luminescence levels detected from droplets comprising the blank and apt-AuNPs. Fig. 5b shows the average luminescence detected from blank droplets (grey circle. 5 samples). The luminescence was statistically different than CL obtained from H-LYS (black spheres. Two-tailed t-test, level 95%, ν = 8, p < 0.0001). We estimated the detection limit of the assay by using the fourparameter logistic curve fit procedure elsewhere described (Holstein et al., 2015). In particular, we determined the limit of blank (LOB) by using the following Eq. (1):

a) H-LYS + apt-AuNPs C-LYS + apt-AuNPs HSA + apt-AuNPs

80 60 40 20

LOB = µ blank + t (1 − α, n − 1) σblank

0 0

50

100

150

200

250

where µblank is the mean of luminescence intensity for n blank replicates (n = 5 in our case), t(1-α, n-1) is the 1-α percentile of t-distribution (n1 degrees of freedom) and σblank is the standard deviation of the replicates. We obtained LOB = 22.74 (pixel) with α = 0.05. We used the following four-parameters logistic function to fit experimental data:

300

Conc. (fM)

CL intensity (a.u.)

220 200

100

b) H-LYS + apt-AuNPs

a−d

CL =

(

1+

80

40 Aver.Blank+/- 3sBlank

0

50

100

150

200

250

[H − LYS ] +2 b c

)

+d (2)

where CL represents the luminescence intensity at [H-LYS] concentration. Since the sigmoidal shape of the dose-response curve exists only on a log scale, we added 2 to the actual concentration to include the negative control ([H-LYS] = 0) in the fitting procedure, as elsewhere described (Tice et al., 2004). The same number was subtracted after the end of the process. Fig. 6 shows the result of the four-parameters logistic fit (grey line. Adjusted R2 = 0.9955) with the lower and upper 95% prediction limits (dashed grey lines). We estimated both the minimum detectable concentration (MDC) and the reliable detection limit (RDL) as the H-LYS concentrations corresponding to the interpolated intersections of the lower asymptote of the upper 95% prediction limit with the fourparameter logistic fit curve and the lower 95% prediction limit, respectively. Such procedure provided MDC = 15.9 fM and RDL = 44.6 fM. Even if both the LOB value as well as the LOD value estimated according to the method elsewhere described (Tice et al., 2004) (6.8 fM) correspond to an analyte concentration lower than MDC, we choose RDL (44.6 fM) as a conservative estimation of the limit of detection (LOD) of the assay. On this basis, the minimum amount of H-LYS producing a detectable signal from one droplet was 0.89 zeptomoles (droplet volume = 20 nL), while the smaller quantity of H-LYS required for a 1 μL sample was 44.6 zeptomoles. LOD we obtained provided an improved analytical sensitivity

60

20

(1)

300 50000

Conc. (fM) Fig. 5. a) Average referenced CL intensity measured from droplets containing H-LYS and apt-AuNPs (black spheres) and droplets containing HSA (grey spheres) or C-LYS (black triangles) and apt-AuNPs. H-LYS concentrations in the 10 fM − 300 fM range produced discriminated CL signals (two-tailed t-test, level 95%, ν = 8, p < 0.0001). HSA and C-LYS levels in the 10 fM − 300 fM range provided CL signals significantly different than those generated by H-LYS at the same concentration (two-tailed t-test, level 95%, ν = 8, p < 0.0001). Error bars represent the confidence interval of the mean at the 95% level (n = 5). A linear response is found in the 40 fM-300 fM H-LYS concentration range. b) Average un-referenced CL intensity detected from droplets containing H-LYS (10 fM – 50 pM n = 4) and apt-AuNPs (black spheres). We referenced CL signals detected from each sample to the luminescence detected from droplet comprising both blank solutions and apt-AuNPs. The average luminescence generated by the latter droplets is shown (grey circle. n = 5). Error bars show ± 3 sBlank dispersion around the average (%CV < 10). We used a four-parameter logistic function to fit the experimental data (grey line).

provided significantly different average CL intensity values for H-LYS and HSA or C-LYS (two-tailed t-test, level 95%, ν = 8, p < 0.0001). This evidence testifies the critical role played by apt immobilised onto the AuNPs surface. Both H-LYS and C-LYS can adsorb non-specifically onto the AuNPs surface when using bare AuNPs. Under similar circumstances, we observed the luminol CL enhancement for both the investigated systems. apt-AuNPs allowed the preferential adsorption of H-LYS onto the surface of nanoparticles. The evident CL signal enhancement we detected only for H-LYS when using apt-AuNPs enable us to suggest that, among the different mechanisms hypothesised for the AuNPs-mediated luminol CL enhancement (Zhang et al., 2005; Karabchevsky et al., 2016), the plasmonic-mediated antenna effect established when luminol molecules are near to the AuNPs surface is the most probable in our case. In fact, the role of apt-AuNPs as catalyst able to favour the decomposition of oxidising agents is limited by the reduced Au surface accessibility on apt-AuNPs compared to AuNPs due to the presence of the surface immobilised apt molecules. At the same time, far-field phenomena seem not to be important in our case, since only the species preferentially absorbed on the AuNPs surface (H-LYS) can trigger a significant CL enhancement. Fig. 5b shows the average un-referenced CL intensity we recorded

Upper prediction limit (95%)

CL Intensity (a.u.)

200 160

Lower prediction limit (95%)

120 80 40

MCD 15.9 (17.9-2) fM RDL 44.6 (46.6-2) fM

0

10

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[H-LYS] (fM) +2 Fig. 6. Four-parameters logistic fit of the experimental data also shown in Fig. 5b. We added 2 to the actual concentration to include the negative control ([H-LYS] = 0) in the fitting procedure, as described in Ref. Tice et al. (2004). The same number was subtracted after the end of the process. We fit experimental data by using Eq. (2). The best fit (adjusted R2 = 0.995) was obtained with parameters: a = 6.1759; b = 0.5488; c = 367.7928; d = 215.8358.

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environment offers a convenient platform for the management of reduced sample volumes. We used samples in the range of 1 μL and already demonstrated that droplet microfluidic platforms can be operated with sample volumes in the range on 200 nL (Giuffrida et al., 2015). The droplet microfluidic-based assay here described also provides improvements in the time required to perform the assay. CL is detected after about 10 min from the mixing of reagents and the droplet generation. The CL enhancement by AuNPs pushed the detection limit down to LOD = 44.6 fM, thus advancing both commercially available methods as well as new approaches described recently. Different aspects are responsible for the final performance of the assays we performed including the concentration of both luminol and catalyst, the type of catalyst and the nanoparticle-enhancement of the generated chemiluminescence. We used a very high concentration for the luminol (30 mM) and a mixture of K4[Fe(CN)6] and H2O2 as the oxidative and catalytic agents. At the best of our knowledge, much lower concentrations have been used by other authors and only in a few cases a mixture of oxidative and catalytic agents has been used (Jing et al., 2011; Zhang et al., 2005; Wang et al., 2016; Jing et al., 2010). We combined the above mentioned specific experimental conditions with the AuNPs enhancement of the chemiluminescence generated as a consequence of the interaction of luminol with LYS. A KD value of 31 nM has been reported for the aptamer-LYS interaction (Colin Cox and Ellington, 2001) while the number of aptamers immobilized per AuNP can be estimated at the 102 level by considering data reported for similar systems (Demers et al., 2000). We used 11 nM concentrated apt-AuNPs. Therefore, the whole concentration of aptamer molecules can be estimated at the micromolar level. However, it is to consider that a much more complicated system is present within each droplet. An important role is played by the luminol responsible for the generated chemiluminescence. Luminol interacts with the adsorbed H-LYS and benefits of the nanoparticle surface interaction to produce enhanced chemiluminescence. In our case, luminol is available at the 30 mM concentration. Finally, it is to be considered appropriately the enhancement of the chemiluminescence signal provided by AuNPs. As a whole, we believe that the chemiluminescence signal generated by the fM concentrated H-LYS is produced after the adsorption of a low number of at H-LYS molecules on the functionalized nanoparticles. The surface functionalization of AuNPs with a suitable aptamer provided selectivity useful to discriminate between the human lysozyme and a lysozyme showing 60% of similarity (chicken lysozyme). We demonstrated that the method here described can be used to quantify H-LYS in human serum samples. In particular, we proved that H-LYS levels allowed to discriminate the serum sample of healthy donor from serum samples obtained from chronic lymphocytic leukemia patients. To summarize, we achieved the femtomolar sensitivity in H-LYS detection with LOD = 44.6 fM (Fig. 5b), when using 30 mM luminol together with a 1:5 mixture of K4[Fe(CN)6] 2.3 mmol L−1 and H2O2 3% and 11 nM apt-AuNPs. Under such experimental conditions, we identified a 20 fM-50 pM dynamic range with a linear range in the 40 fM300 fM range. We have not been able to discriminate samples with HLYS concentrations higher than 50 pM due to signal saturation. We have been able to discriminate between samples with H-LYS in the nM range when using reduced luminol concentration (10 mM) (Fig. S1). Under such experimental conditions, we observed a linear response in the 5–100 nM H-LYS concentration range. The experiments we conducted by spiking 1:100 diluted human serum samples with 10 nM, 15 nM and 20 nM H-LYS confirmed the presence of a linear response in the investigated H-LYS concentration range (Fig. S2). Considering the low background luminescence generated in the absence of the target protein we could hypothesise that the described method could have an application also in digital detection. Different experimental configurations should be investigated to prove the real transferability of the proposed approach to the digital detection. However, the evidence presented in this paper represents a good

compared to other methods recently described. In particular, it is lower than those achieved with an impedimetric aptasensor based on carbon nanotubes-modified screen-printed electrodes (LOD = 862 nM) (Rohrbach et al., 2012), with the CdTe/CdSe quantum dot fluorescence detection combined with a rolling circle amplification (LOD = 2.6 nM) (Qiu et al., 2017) and with a graphene-based SPR aptasensor (LOD = 0.5 nM) (Subramanian et al., 2013). The droplet microfluidics method here described competes with standard ELISA assays for H-LYS detection. It allows overcoming issues affecting ELISA assays and arising from the potential cross-reactivity of secondary antibodies responsible for the generation of non-specific signals. When compared with commercially available ELISA kits, whose limit of detection is in the 0.45 femtomoles range (LOD = 9 pM, 50 μL sample volume) (〈http://www.abcam.com/human-lysozyme-elisa-kitlzm-ab108880-references.html〉) our method achieved a better LOD in the range of 44.6 zeptomoles. The detection of H-LYS here described is also competitive with commercially available kits also regarding the analysis time (about 20 min from sample loading and signal detection). 3.4. Detection of lysozyme in human serum samples To demonstrate the applicability of the described method into the clinical practice, we used the droplet microfluidic-based assay to detect H-LYS in human serum samples obtained from selected in-patients of the "Regina Elena National Cancer Institute". We selected the serum samples considering the total white blood cell count (WBC) and classified the samples as ‘normal’ (sample ID 10265267, WBC 7900; sample ID 10250120, WBC 5400) or ‘pathological’ (sample ID 10265574, WBC 113000; sample ID 10265945, WBC 63000 and sample ID 10255837, WBC 165000) accordingly. In particular, we obtained pathological serum samples from patients with chronic lymphocytic leukemia or medullary acute leukemia (sample ID 10265945). To verify the applicability of the method, we fortified 1:100 diluted human serum samples from one healthy donor (sample ID 10265267) and two chronic lymphocytic leukemia patients (sample ID 10265574 and 10255837) with 1 pM concentration of H-LYS and analysed the fortified samples under the same experimental conditions used to obtain data shown in Figs. 5 and 6. After the droplet-microfluidic-based analysis we calculated percentage recovery values of 94.9%, 86% and 91% for the three serum samples, considering the H-LYS concentration found in the fortified serum sample and H-LYS concentration found in the same samples before fortification. Then we fortified 1:100 diluted human serum samples with a higher H-LYS concentration than before (50 nM) to evaluate the assay ability to properly operate also with high H-LYS concentrations. We operated by using 10 mmol L−1 luminol in NaOH 0.5 mol L−1 and a 1:10 mixture of K4[Fe(CN)6] 2.3 mmol L−1 and H2O2 3%. Also in this case, after the droplet-microfluidic-based analysis we calculated percentage recovery values of 98% (sample ID 10250120), 95.36% (sample ID 10265945) and 97.44% (sample ID 10255837) by using the calibration curve shown in Fig. S1 (supplementary material). H-LYS concentration in the latter three 1:100 diluted serum samples was determined by using the method of standard addition (Fig. S2). Concentration of H-LYS found was 58.6 nM for sample ID 10250120, 563 nM for sample ID 10265945 and 1755 nM for sample ID 10255837. Such values correlated with the white blood cells count (5400, 63000 and 165000, respectively) and allowed to discriminate the healthy donor from chronic lymphocytic leukemia and medullary acute leukemia patients. 4. Conclusions This study demonstrates that the combined use of droplet microfluidics, AuNPs-enhanced chemiluminescence and surface modification of AuNPs with an anti-H-LYS aptamer is an attractive alternative for the construction of a sensitive H-LYS biosensing platform to be used for HLYS detection in human serum samples. The droplet microfluidics 13

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starting point for the possible development of a digital detection assay.

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