Annette Delices

Davina Moodelly

Charlotte Hurot

Yanxia Hou

Wai Li

Christine Saint-Pierre

Didier Gasparutto

Gilles Nogues

Peter Reiss

Kuntheak Kheng

Aqueous synthesis of DNA-Functionalized Near-Infrared AgInS 2 /ZnS Core/Shell Quantum Dots

Keywords: Quantum dots, semiconductor nanocrystals, AgInS2, core-shell, hot-injection, seed-mediated growth, DNA functionalization, surface plasmon resonance

ABSTRACT

The unique photophysical properties of quantum dots (QDs) paved the way for the development of advanced emitting nanomaterials in bio-imaging and biosensing applications.

Biocompatibility, biofunctionality, non-toxicity as well as colloidal and chemical stability are essential criteria to be fulfilled by the QD emitters. At present, it remains challenging to achieve efficient near-infrared emitting QDs respecting these criteria, in particular in the absence of toxic heavy metals such as cadmium or lead. In this perspective, we developed water soluble nearinfrared emitting AgInS2/ZnS core/shell (AIS/ZnS) QDs functionalized with DNA. The newly established aqueous route relying on a two-step hot-injection synthesis led to highly luminescent chalcopyrite-type AIS/ZnS core/shell QDs with an unprecedented photoluminescence quantum yield (PLQY) of 55% at 700 nm and a long photoluminescence decay time of 900 ns. Fast and slow hot-injection of the precursors were compared for the AIS core QDs synthesis, yielding a completely different behavior in terms of size, size distribution, stoichiometry and crystal structure. The PL peak positions of both types of core QDs were 710 (fast) and 760 nm (slow injection) with PLQYs of 36% and 8%, respectively. The slow and successive incorporation of the Zn and S precursors during the subsequent shell growth step on the stronger emitting cores promoted the formation of a three-monolayer thick ZnS shell, evidenced by the increase of the average QD size from 3.0 nm to 4.8 nm. Bioconjugation of the AIS/ZnS QDs with hexylthiol modified DNA was achieved during the ZnS shell growth, resulting in a grafting level of 5-6 DNA single strands per QD. The successful chemical conjugation of DNA was attested by UV-Vis spectroscopy and agarose gel electrophoresis. Importantly, surface plasmon resonance imaging (SPRi) experiments using complementary DNA strands further corroborated the successful coupling and the stability of the AIS/ZnS-DNA QD conjugates as well as the preservation of the biological activity of the anchored DNA. The strong NIR emission, long-term stability and biocompatibility of the obtained AIS/ZnS-DNA QDs provide a high potential for their use in biomedical applications.

INTRODUCTION

Significant progress has been made in the development of advanced emitting nanomaterials including nanoscale metal organic frameworks, carbon nanotubes, carbon dots and quantum dots (QDs), as alternatives to conventional organic fluorophores for biosensing and bio-imaging applications. [START_REF] Park | Size-Controlled Synthesis of Porphyrinic Metal-Organic Framework and Functionalization for Targeted Photodynamic Therapy[END_REF][START_REF] Iverson | In Vivo Biosensing via Tissue-Localizable near-Infrared-Fluorescent Single-Walled Carbon Nanotubes[END_REF][START_REF] Jiang | Blue Luminescence by Carbon Dots: Full-Color Emission Tuning and Multicolor Cellular Imaging[END_REF][START_REF] Lv | Near-Infrared Emission CuInS/ZnS Dots: All-in-One Theranostic Nanomedicines with Intrinsic Fluorescence/Photoacoustic Imaging for Tumor Phototherapy[END_REF] For such applications, near-infrared (NIR) emitters are of particular interest to minimize autofluorescence as well as light absorption and scattering from the biological environment. QDs have attracted huge attention in this context, owing to their unique photophysical properties, specifically their photoluminescence (PL) at tunable wavelength across the visible, NIR-I and NIR-II regions determined by their size, shape and chemical composition. [START_REF] Kovalenko | Prospects of Nanoscience with Nanocrystals[END_REF][START_REF] Zhao | Near Infrared Quantum Dots in Biomedical Applications: Current Status and Future Perspective: Near Infrared Quantum Dots in Biomedical Applications[END_REF] Furthermore, their high quantum yield (PLQY), long PL lifetime, long-term stability and strong photobleaching resistance, place QDs among the most attractive fluorescent nanomaterials for bio-applications. However, commonly used binary NIR QDs exhibit (cyto-)toxic effects caused by the presence of hazardous elements (e.g., Cd, Pb). Therefore, growing efforts are devoted to the development of biocompatible and toxic heavy metal-free QDs. [START_REF] Reiss | Synthesis of Semiconductor Nanocrystals, Focusing on Nontoxic and Earth-Abundant Materials[END_REF][START_REF] Zhang | High-Efficiency Green InP Quantum Dot-Based Electroluminescent Device Comprising Thick-Shell Quantum Dots[END_REF][START_REF] Bai | Hydroxyl-Terminated CuInS Based Quantum Dots: Toward Efficient and Bright Light Emitting Diodes[END_REF][START_REF] Tsolekile | Fluorescence Tagging and Gene-Expression Study of CuInS/ZnS QDS -Meso (Hydroxyphenyl) Porphyrin Conjugate against Human Monocytic Leukemia Cells[END_REF][START_REF] Zeng | Interfacial Synthesis of Ag 2 S/ZnS Core/Shell Quantum Dots in a Droplet Microreactor[END_REF] Among them, ternary I-III-VI chalcopyrite-type materials are becoming the focus of interest. [START_REF] Aldakov | Ternary and Quaternary Metal Chalcogenide Nanocrystals: Synthesis, Properties and Applications[END_REF][START_REF] Zhong | Tuning the Luminescence Properties of Colloidal I-III-VI Semiconductor Nanocrystals for Optoelectronics and Biotechnology Applications[END_REF][START_REF] Girma | Synthetic Strategies and Biomedical Applications of I-III-VI Ternary Quantum Dots[END_REF] These ternary QDs, regarded as promising eco-friendly materials for multiple applications, display a direct band gap (1.04-1.87 eV in the bulk materials) and high absorption coefficients (10 4 -10 5 cm -1 ). Among them, silver indium sulfide (AgInS2, AIS) QDs, and their alloys with Zn, stand out from their analogues thanks to their elevated PLQY and long lifetime (several hundred nanoseconds) regardless of the synthetic medium (organics or water). Nevertheless, AIS QDs prepared in organic solvents generally demonstrate better photophysical properties [START_REF] Ko | Highly Efficient Green Zn-Ag-In-S/Zn-In-S/ZnS QDs by a Strong Exothermic Reaction for Down-Converted Green and Tripackage White LEDs[END_REF] but are not directly applicable in biological systems. Aqueous phase transfer via ligand exchange is feasible but is known to result in a significant loss of PLQY. [START_REF] Li | Highly Luminescent CuInS 2 /ZnS Core/Shell Nanocrystals: Cadmium-Free Quantum Dots for In Vivo Imaging[END_REF] Therefore, aqueous synthesis is of particular interest to directly obtain water-soluble AIS QDs. To date, in aqueous medium, several colloidal synthetic approaches have been reported such as heat-up, hot-injection, hydrothermal and microwave irradiation assisted methods. [START_REF] Girma | Synthetic Strategies and Biomedical Applications of I-III-VI Ternary Quantum Dots[END_REF][START_REF] Moodelly | Photophysical Properties and Surface Chemistry of Chalcopyrite-Type Semiconductor Nanocrystals[END_REF][START_REF] Jain | I-III-VI Core/Shell QDs: Synthesis, Characterizations and Applications[END_REF] The brightest AIS QDs have been produced by heat-up (PLQY ~60 % [START_REF] Stroyuk | Origin and Dynamics of Highly Efficient Broadband Photoluminescence of Aqueous Glutathione-Capped Size-Selected Ag-In-S Quantum Dots[END_REF][START_REF] Mrad | Aqueous Synthesis for Highly Emissive 3-Mercaptopropionic Acid-Capped AIZS Quantum Dots[END_REF] ) and microwave (PLQY ~66 % [START_REF] Martynenko | Photoluminescence of Ag-In-S/ZnS Quantum Dots: Excitation Energy Dependence and Low-Energy Electronic Structure[END_REF] ) approaches. To make them compatible with biomedical applications, generally further surface chemical modification is required. [START_REF] Bruchez | Semiconductor Nanocrystals as Fluorescent Biological Labels[END_REF] and proteins [START_REF] Chan | Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection[END_REF] , respectively, in combination with CdSe/ZnS core/shell QDs. The following year, Mirkin et al. initiated the functionalization of QDs with single stranded DNA (ssDNA) bound by a thiol functional group. [START_REF] Mitchell | Programmed Assembly of DNA Functionalized Quantum Dots[END_REF] Since then, a large number of advanced hybrid-QD nanostructures using DNA have been elaborated, constituting a major breakthrough in biomedical applications. Several pathways are possible to functionalize QDs with DNA. For example, Sun and Gang achieved covalent bonding by coupling carboxyl groups on the QD surface with amino-DNA. [START_REF] Sun | DNA-Functionalized Quantum Dots: Fabrication, Structural, and Physicochemical Properties[END_REF] Along the same line, the group of Dubertret triggered the reaction between amino-DNA with thiol functions present on an amphiphilic polymer used to encapsulate the QDs. [START_REF] Banerjee | Efficient, and Stable Conjugation of Multiple DNA Strands on Colloidal Quantum Dots[END_REF] In both studies, the resulting QDs are structurally and optically stable while varying the pH and the ionic strength. Another approach is the direct attachment of ssDNA during the inorganic (ZnS, CdS) shell growth. This one-step method relies on the introduction of thiol or phosphorothioate modified ssDNA in the shell precursors' solution which acts as capping and stabilizing agent. [START_REF] Wang | Quantum Dot Bioconjugation during Core-Shell Synthesis[END_REF][START_REF] Samanta | Infrared Emitting Quantum Dots: DNA Conjugation and DNA Origami Directed Self-Assembly[END_REF][START_REF] Samanta | Robust DNA-Functionalized Core/Shell Quantum Dots with Fluorescent Emission Spanning from UV-Vis to Near-IR and Compatible with DNA-Directed Self-Assembly[END_REF][START_REF] Tikhomirov | DNA-Based Programming of Quantum Dot Valency, Self-Assembly and Luminescence[END_REF][START_REF] Zhang | DNA-Based Assembly of Quantum Dots into Dimers and Helices[END_REF] All these works were carried out using CdSe, CdTe and CdxPb1-xTe QDs and so far, no study has been devoted to the DNA-functionalization of ternary QDs such as AIS.

Herein, we report a novel synthesis method for AIS/ZnS core/shell QDs via a two-step procedure as well as an efficient way for their surface functionalization with ssDNA while preserving their strong luminescence. First, the aqueous synthesis of NIR-emitting AIS core QDs (cQDs) was performed using two different approaches, by comparing the classical fast hot-injection method with a yet unexplored slow hot-injection procedure referring to seed-mediated growth. Both methods led to significant differences in the QD properties, yielding 3.0 nm Ag-poor Ag0.53InS2

QDs crystallizing in the chalcopyrite phase (fast injection) and 5.2 nm close-to-stoichiometric Ag1.1InS2.1 QDs of lower size distribution, crystallizing in the orthorhombic phase (slow injection). The PL peak positions of two types of QDs were 710 and 760 nm with PLQYs of 36% and 8%, respectively. Capping the stronger emitting cQDs with a ZnS shell layer led to highly luminescent AIS/ZnS core/shell QDs (csQDs) reaching a PLQY of 55%, stabilized in the aqueous phase by hydrophilic glutathione (GSH) ligands. Up to now, producing a thick ZnS shell layer on AIS QDs was challenging in aqueous medium. Here we adopted an approach based on the slow and successive incorporation of Zn and S precursor solutions promoting the formation of a thick shell evidenced by a QD size increase from 3.0 nm to 4.8 nm. Hexylthiol modified ssDNA was incorporated during the ZnS shell growth under precise control of pH and ionic strength. Successful chemical conjugation of ssDNA affording csQD-DNA was attested by several techniques (UV-Vis spectroscopy, agarose gel electrophoresis). Importantly, surface plasmon resonance imaging (SPRi) experiments using complementary DNA strands further corroborated the successful coupling and the stability of the bright AIS csQD-DNA conjugates as well as the preservation of the biological activity of the anchored DNA.

EXPERIMENTAL SECTION

Materials

All the chemicals were used as received without further purification. Silver nitrate (AgNO3, 99.99 %), indium (III) chloride (InCl3, 99.99 %), sodium sulfide nonahydrate (Na2S.9H2O), Lglutathione reduced (GSH), ammonium citrate dibasic (C6H6O7.2NH4, 98 %), zinc acetate dihydrate (Zn(CH3COO)2.2H2O, 99.99 %), poly(ethylene glycol) methyl ether thiol (PEG-SH, Mn= 2000 Da) phosphate buffered saline tablet (PBS), sodium chloride (NaCl, 99 %), sodium hydroxide pellet (NaOH, 98 %), potassium phosphate dibasic (K2HPO4, 98%) and glycerol (99%) were all purchased from Sigma Aldrich. 2-Propanol (99.8 %) and ammonium hydroxide solution (NH4OH, 33 %) were supplied by Fisher Scientific. The thiol (HS-(CH2)11-(EG)3-OH, Mw= 336 Da) is purchased from Prochimia. MilliQ ultrapure water (18.2 MΩ.cm) was used in all experiments. All the modified oligonucleotides were customized and purified by Eurogentec, the sequences are listed below: 5' GTGCGAGTGAATTCCGAAGGTATTT-SH 3' (ssDNAQD; ε= 247800 M -1 .cm -1 ) 5' TACCTTCGGAATTCACTCGCACTTTTTTTTTTTT-SH 3' (ssDNASPRcc; ε= 297300 M - 1 .cm -1 ) 5' GACCATCGTGCGGGTAGGTAGACCTTTTTTTTTTTT-SH 3' (ssDNASPRcontrol; ε= 332500

M -1 .cm -1 )
AIS core synthesis (cQDs) cQDs were synthesized via hot-injection synthetic method in two different ways: the "fast" and "slow" injection.

 Fast hot-injection approach

Typically, 8 mL of 0.1 M GSH and 70 mg of ammonium citrate were added into a three-neck flask. The pH was adjusted to 6.5 using NH4OH (5 M). Subsequently, 0.5 mL of 1 M InCl3 was introduced, leading to the formation of a precipitate. Then NH4OH was added until complete dissolution of the precipitate and the pH of the mixture increases back to 6.5.. The reaction mixture was brought to boiling and then first 1.25 mL of 0.1 M AgNO3 and next 625 µL of 1 M Na2S were quickly injected. The solution was kept boiling under stirring for 1h. The color of the solution changed from transparent to brownish red.

 Slow hot-injection approach

The procedure is similar to the fast injection approach except that the addition of AgNO3 and Na2S is different. Briefly, once the mixture containing GSH, ammonium citrate, and InCl3 was brought to boiling, 250 µL of 0.1 M AgNO3 and 125 µL of 1 M Na2S were quickly injected to form the AIS seeds. Then, 1 mL of 0.1 M AgNO3 and 500 µL of 1 M Na2S were successively added dropwise using a syringe pump for 20 min. The solution was kept boiling under stirring for 1h. The color of the solution was brownish red but darker than the fast injection approach.

AIS/ZnS core/shell synthesis (csQDs)

For the ZnS shell growth on cQDs, two distinct solutions were prepared (A and B). Solution A contained 7 mL of Zn(CH3COO)2.2H2O stock solution (0.1 M) and 7 mL of the stabilizing ligand GSH (0.1 M) completed with 2 mL H2O. Solution B contained 700 µL of Na2S (1 M) and 15.3 mL H2O. Both solutions were added in parallel and slowly via a syringe pump with a constant flow rate of 0.15 mL/min into the core solution (V= 4 mL) at constant temperature (96 °C). Two aliquots were extracted from the solution after 50 and 100 min during the addition, respectively, for characterization in order to follow the growth process. After completed injection, the mixture was maintained at 96 °C for another hour.

DNA Functionalization on AIS/ZnS QDs (csQD-DNA) DNA single strands were attached to the ZnS shell during its synthesis. Briefly, 100 µL of the cQDs solution diluted in 5 mL of H2O was heated to 96 °C. Then, 2 mL of an aqueous solution containing 97 µL of Zn(CH3COO)2.2H2O stock solution (0.1 M) and 1 mL of HS-DNA (ssDNAQD ; C= 0.1 M in PBS at pH 7.4) and 2 mL of another solution containing 9.7 µL Na2S (1 M) were added dropwise and in parallel via a syringe pump. After the addition, the mixture was maintained at this temperature for 1h. For better comparison, csQDs without DNA were prepared under the same condition except that DNA was replaced by GSH.

QDs purification

All QDs were purified by precipitation performed by the addition of 2-propanol followed by centrifugation (10303 g for 5 min). The pellet was redissolved in H2O. For csQD-DNA, they were purified by centrifugation through vivaspin centrifugal filter with a 300 kDa molecular weight cut off (3 times centrifugation at 10303 g for 2 min). The photoluminescence decay curves were recorded using the time-correlated single-photon counting (TCSPC) technique on Fluorolog-3 spectrometer (Horiba Jobin Yvon) and the excitation source was a pulsed light emitting diodes with an excitation wavelength of 455 nm (NanoLED-05A, >1.3 ns pulse, Horiba). The curves were fitted with DAS6 analysis software provided by Horiba Scientific. For measurements of photoluminescence quantum yields (PLQY) a G8 integrated sphere module (GMP SA, Switzerland) was used in the Fluorolog-3 spectrometer (Horiba Jobin Yvon).

Characterization Techniques

Agarose gel electrophoresis

To qualitatively attest the presence of DNA on QDs, csQD-DNA conjugates were characterized by fluorescence detection in agarose gel electrophoresis. cQDs and csQDs were used as control. Each solution contains 8 µL of the corresponding sample, 2 µL of the loading dye and 0.5 µL of the DNA intercalating agent (SYBR Gold) for some of them. The migration was performed with a FlashGelTM System (Lonza) using a FlashgelTM DNA cassette (1.2% agarose gel) and was running at 120 V.

Surface plasmon resonance imaging (SPRi)

Surface plasmon resonance imaging was used to confirm the presence of DNA on the QDs after the functionalization, and most importantly, to evaluate the biological activities of the DNA ligands on QDs. For this, first, a biochip was prepared using the complementary DNA strand (ssDNASPRcc) and an arbitrary DNA strand (ssDNASPRcontrol) for negative control. They were immobilized onto a SPRi prism coated with a thin gold film supplied by Horiba Scientific. 48h before use, the gold surface of the prism was subjected to plasma treatment with a Femto plasma cleaner (Diener electronic) (25 % argon, 75 % oxygen, 0.6 mbar, 40 W, 3 min). Then, droplets of DNA solutions were deposited on the prism by a non-contact spotting robot (sciFLEXARRAYER S3, Scienion, Germany). In this study, DNA spotting solutions were prepared in 4 different buffer solutions for optimization: 1) PBS with pH=7.4; 2) 0.1 M K2HPO4 with pH= 9.25; 3) 0.5 M K2HPO4 with pH= 9.25; 4) 1 M K2HPO4 with pH= 9.25. Each spotting solution contains 10 µM of DNA, 10 µM of PEG-SH (Mn= 2000 Da) and 5 % glycerol in the corresponding buffer. Moreover, for each condition, three replicates were randomly deposited on the prism to avoid any bias due to the position effect on the biochip. After spotting, the prism was placed in a humidity-controlled chamber (relative humidity of 96%) at 25 °C for 24h for the immobilization of DNA by self-assembly. Then, the prism was rinsed thoroughly with deionized water. Finally, the free gold surface on the prism was blocked with PEG-SH (Mw=336 Da, 100 µM) for 1h and rinsed completely with deionized water.

The biochip was then used for the analysis of csQD-DNA conjugates using the classical Kretschmann-type optical configuration at a fixed working angle. The measurements were carried out with an OpenPleX system (Horiba Scientific) placed in an incubator at 25 °C. For this, a collimated, polarized light beam at 635 nm was sent through the prism to illuminate the whole biochip. The reflectivity of all the DNA spots was simultaneously monitored and recorded with a CCD camera, with each pixel mapping a specific position on the prism. Upon the injection of csQD-DNA conjugates, binding events occurred. Consequently, the reflectivity of the spots on the biochip increased, which was recorded. A measurement was taken every three seconds.

Thanks to image treatment software, SPR images were then converted to variations of reflectivity (expressed as Δ Reflectivity, %) versus time, generating a series of kinetic binding curves, called sensorgrams.

In practice, the prism was mounted into a 10 μL PEEK flow cell, which was connected to a fluidic system. Filtered running buffer (PBS at pH 7.4 with 0.4 M NaCl) was pushed by a computer-controlled syringe pump (Cavro Scientific Instruments, USA) at a constant flow of 0.52 μL/s. In addition, it passed through a degassing system (Alltech, France) before arrival in the flow cell to eliminate air bubbles. The samples of csQD, ssDNAQD and csQD-DNA conjugates were manually injected through a six-port injection valve (Upchurch Sci., USA) using a syringe. The injection loop volume was 500 µL. After each analysis, the biochip was completely regenerated in situ with 0.2 M NaOH for re-use. csQD-DNA were used for the SPR measurements few days to more than 3 months after their preparation. In this work, only the results obtained after 3 months are illustrated.

RESULTS AND DISCUSSION

AIS core QDs synthesis: fast vs. slow hot-injection

In this study, a two-step procedure in aqueous medium has been developed to synthesize AIS/ZnS core/shell QDs functionalized with DNA, as depicted in Scheme 1. In the first step, the preparation of the core QDs (cQDs) used the same chemicals as in the synthesis reported by Stroyuk et al. [START_REF] Stroyuk | Origin and Dynamics of Highly Efficient Broadband Photoluminescence of Aqueous Glutathione-Capped Size-Selected Ag-In-S Quantum Dots[END_REF] Briefly, AgNO3, InCl3 and Na2S were brought to react together with a molar ratio set at 1:4:5 and GSH and citrate were added as stabilizing agents. However, the reported heat-up synthesis led to a very broad size distribution of the QDs, requiring supplementary post-synthetic size fractionation steps. Furthermore, it is challenging to access the NIR range with emission peaks centered at wavelengths exceeding 650 nm. To overcome these limitations, we explored a different synthetic approach applying a hot-injection method. Typically, this approach involves a fast injection of one or several precursors into a hot reaction mixture limited by the solvent boiling temperature. [START_REF] Murray | Synthesis and Characterization of Nearly Monodisperse CdE (E = Sulfur, Selenium, Tellurium) Semiconductor Nanocrystallites[END_REF][START_REF] Peng | Kinetics of II-VI and III-V Colloidal Semiconductor Nanocrystal Growth: "Focusing" of Size Distributions[END_REF] In aqueous medium, the boiling temperature is considerably lower compared to organic media and thus does not provide the same degree of freedom for modulating the nucleation and growth temperatures. However, this approach could lead to nearly monodispersed AIS QDs showing a maximum PLQY of up to 3% and 15 % for AIS/ZnS QDs [START_REF] Luo | One-Step Synthesis of Water-Soluble AgInS2 and ZnS-AgInS2 Composite Nanocrystals and Their Photocatalytic Activities[END_REF] with a core synthesis using AgOAc, In(OAc)3, Na2S and GSH. To optimize the overall PLQY and the uniformity in size and shape of the cQDs two methods have been compared. The first one is the classical fast hot-injection of precursors while the second one applies slow hotinjection, referred to as seed-mediated growth.

Among the soluble metal salt used in the preparation of AIS QDs, AgNO3 and InCl3 have been chosen as silver and indium sources, respectively. At pH >3.5, In 3+ is no longer complexed by Cl ¯ but with HO ¯ and must precipitate. For this reason, the prior presence of citrate as chelating agent in solution is necessary to prevent the precipitation of indium. The kinetic of indium citrate formation is faster than the indium hydroxide complex formation. And compared to that of indium acetate, the stability constant is higher for the indium citrate complex. Since InCl3 is a strong Lewis acid, it will react with the strongest base present in the solution i.e. citrate, on the basis of Pearson's hard and soft acids and bases (HSAB) principle. [START_REF] Pearson | Hard and Soft Acids and Bases[END_REF] As for AgNO3, the soft acid Ag + will interact with the sulfhydryl group of GSH to form a digonal coordination complex chain (-S(G)-Ag-S(G)-), [START_REF] Bell | Structural Chemistry and Geochemistry of Silver-Sulfur Compounds: Critical Review[END_REF][START_REF] Leung | Silver(I) Complex Formation with Cysteine, Penicillamine, and Glutathione[END_REF] leading to a pale yellow solution. Immediately after Na2S injection the solution turns to dark brown indicating that HS¯ has reacted with silver thiolate to form intermediate Ag2S. [START_REF] Bell | Structural Chemistry and Geochemistry of Silver-Sulfur Compounds: Critical Review[END_REF] Right after the reaction mixture becomes brownish red, which is the typical color of AgInS2 QDs formed by cation exchange with In 3+ (2 Ag2S + In 3+ → AgInS2 + 3 Ag + ). [START_REF] Song | Bandgap and Structure Engineering via Cation Exchange: From Binary Ag 2 S to Ternary AgInS 2 , Quaternary AgZnInS Alloy and AgZnInS/ZnS Core/Shell Fluorescent Nanocrystals for Bioimaging[END_REF] As an alternative approach aiming at reducing the size distribution of the cQDs, we explored here the seed-mediated growth mechanism. This approach is widely applied for the preparation of larger noble metal nanoparticles, magnetic nanocrystals and QDs synthesized in organic solvents. Here, in contrast to the fast injection where the AgNO3 and Na2S precursors are completely added at once, a slow injection is applied to ensure the progressive, continuous and uniform growth of the same material as the preformed seeds. The synthesis is performed at the boiling point (100°C) which induces the rapid decomposition of the injected precursors into multiple monomers that deposit on the seeds surface leading to the formation of larger QDs. The solution aspect during the synthesis is similar to that of the previous synthesis prior to the seed formation. Then, the solution gets increasingly dark, indicating the growth of the QDs.

The cQDs morphology and the size distribution were analyzed by TEM and the images are shown in Figure 1. In general, regardless of the approach used for the synthesis, the particles are fairly uniform with a well-defined spherical shape. In the case of slow precursor injection, the particles are bigger, with an average size diameter of 5.2 ± 0.6 nm and a narrow size distribution of 11.5 %. The QD solution appears darker than in the case of the "fast injection", which leads to a significantly smaller size of 3.0 ± 0.6 nm and larger size distribution (20 %). The chemical composition was determined by EDX. It was found that for cQDs obtained with the fast precursor injection, a nonstoichiometric composition of Ag:In:S= 1:1.9:3.8 is obtained showing a strongly silver-deficient composition (Ag0.53InS2). This indicates that silver vacancies (VAg) are present within the crystal lattice, which act as acceptor type defects. To the contrast, the cQDs obtained with the slow precursor injection are more Ag-enriched showing a quasi-stoichiometric composition of Ag:In:S= 1:0.91:1.9 (Ag1.1InS2.1). For both syntheses, it should be noted that the initial Ag:In:S ratio was set at 1:4:5, considered as the optimum ratio to yield the highest PL intensity based on literature. [START_REF] Stroyuk | Origin and Dynamics of Highly Efficient Broadband Photoluminescence of Aqueous Glutathione-Capped Size-Selected Ag-In-S Quantum Dots[END_REF][START_REF] Martynenko | Photoluminescence of Ag-In-S/ZnS Quantum Dots: Excitation Energy Dependence and Low-Energy Electronic Structure[END_REF] Comparing with the elemental analyses suggests that the cQDs composition strongly depends on the reaction kinetics and the unbalanced precursors' reactivity.

For the fast-injection, the Ag-deficient cQDs composition and the Ag:In= 0.53:1 cationic ratio were expected as the starting ratio was Ag:In=1:4 and In 3+ has lower reactivity than Ag + . In the case of the quasi-stoichiometric composition, the slow injection of Ag and S favors the growth of the seeds but minimizes the cation exchange reaction between In 3+ and Ag + which explains the larger Ag content in these cQDs. The expected defects in this case are in the form of silver (AgInt) and sulfur (SInt) interstitials acting as donors and acceptors, respectively. Using powder X-Ray diffraction (Figure 2b) the cQDs obtained with the two different synthetic routes can clearly be distinguished. For the "fast injection" cQDs, the diffractogram shows mainly three broad peaks centered at 27.1°, 45.3° and 52.9° that match well with the (112), ( 204) and (312) lattice planes of the tetragonal chalcopyrite reference pattern (JCPDS 00-025-1330). As for the "slow injection" cQDs, an orthorhombic phase is predominantly observed (JCPDS 00-025-1328) with two broader peaks at 28.3° and 48° with a small shoulder at 52.7° associated to the (121), (320), (322) lattice planes respectively. The broader diffraction peaks indicate that the stoichiometric cQDs have a poorer crystallinity.

As shown in Figure 2c, both types of cQDs exhibit a broadband PL emission in the NIR-I region. The broad PL bandwidth is a specific feature of ternary I-III-IV2 QDs ascribed to transitions between photoexcited electrons delocalized in the conduction band and holes localized at an acceptor-type defect level within the band gap, referred to as the free-to-bound transition. [START_REF] Moodelly | Photophysical Properties and Surface Chemistry of Chalcopyrite-Type Semiconductor Nanocrystals[END_REF][START_REF] Berends | Optoelectronic Properties of Ternary I-III-VI 2 Semiconductor Nanocrystals: Bright Prospects with Elusive Origins[END_REF] However, we note that the line width of the obtained QDs (around 300 meV) is significantly narrower than that obtained with established heat-up syntheses. [START_REF] Moodelly | Photophysical Properties and Surface Chemistry of Chalcopyrite-Type Semiconductor Nanocrystals[END_REF] The PL band centered at 760 nm that corresponds to the "slow injection" cQDs, is weakly photoluminescent (PLQY= 8 %). In contrast, the "fast injection" cQDs exhibit a PL band at shorter wavelength (710 nm) due to Ag-deficient composition, with significantly stronger photoluminescence (PLQY= 36 %). The higher PLQY can be attributed to the higher crystallinity and to the higher density of defect states in the Ag-deficient cQDs. [START_REF] Moodelly | Photophysical Properties and Surface Chemistry of Chalcopyrite-Type Semiconductor Nanocrystals[END_REF] In the absorption spectra (Fig. 2d) no apparent excitonic peak is visible but only a large shoulder centered at 420 nm for the "fast injection" cQDs and at 550 nm for "slow injection" cQDs. To get further insight on the optical properties, time-resolved photoluminescence measurements have been performed to determine the photoluminescence lifetime of the QDs (Fig. 2e). As most of the ternary QDs, the decay curves were fitted by a multi-exponential function [START_REF] Sun | Photocarrier Recombination Dynamics in Ternary Chalcogenide CuInS 2 Quantum Dots[END_REF][START_REF] Park | Photoluminescence of Band Gap States in AgInS 2 Nanoparticles[END_REF][START_REF] Berends | Radiative and Nonradiative Recombination in CuInS 2 Nanocrystals and CuInS 2 -Based Core/Shell Nanocrystals[END_REF][START_REF] Zhong | Controlled Synthesis and Optical Properties of Colloidal Ternary Chalcogenide CuInS 2 Nanocrystals[END_REF][START_REF] Li | Efficient Synthesis of Highly Luminescent Copper Indium Sulfide-Based Core/Shell Nanocrystals with Surprisingly Long-Lived Emission[END_REF][START_REF] Mir | Bandgap Tunable AgInS Based Quantum Dots for High Contrast Cell Imaging with Enhanced Photodynamic and Antifungal Applications[END_REF] . For both cQDs, the decay curve displays a triple exponential model considering the chi-square values (χ²) and the accurate fit in the short time (Fig. S1 and Table S1):

𝐼(𝑡) = 𝐴 𝑒 + 𝐴 𝑒 + 𝐴 𝑒

where τ is the decay time and A is the relative amplitude of each decay component. The three components are attributed to three different radiative recombination processes. The first decay time τ1 refers to the fast photo-carrier recombination process on the QD surface defects mainly caused by dangling bonds, τ2 to the recombination process on QD surface states and the last decay time τ3 to recombination inside the core states [START_REF] Berends | Optoelectronic Properties of Ternary I-III-VI 2 Semiconductor Nanocrystals: Bright Prospects with Elusive Origins[END_REF][START_REF] Chevallier | Photoluminescence Properties of AgInS 2 -ZnS Nanocrystals: The Critical Role of the Surface[END_REF] (Table 1). The "fast injection" cQDs show a long fluorescence lifetime of more than 990 ns, with a contribution of 77 % of the total emission. Therefore, the radiative recombination process within the QD core mainly dominates the fluorescence emission similarly to the "slow injection" cQDs where the contribution is comparable (86 %), again with a long lifetime of 810 ns.

To conclude this part, AIS core QDs have been successfully prepared according to two synthetic approaches. Although the seed-mediated growth route has led to larger, close-tostoichiometric, orthorhombic particles of lower size distribution, the chalcopyrite-type QDs resulting from the classical fast hot-injection route exhibit a significantly higher PLQY of (36 % vs. 8 %) with a longer lifetime exceeding 900 ns which are beneficial for bio-applications.

Therefore, for the following surface engineering steps the chalcopyrite-type cQDs are considered

AIS/ZnS core/shell QDs

To protect the AIS core QDs against photodegradation and to further enhance their PLQY, a ZnS shell was grown in a second step of the synthesis. Ubiquitously used, ZnS is a wide band gap semiconductor (3.7 eV) showing a low lattice mismatch with AIS cQDs (8 %). Different routes for ZnS shell growth on AIS QDs in the aqueous phase have been reported in literature including thermal decomposition [START_REF] Raevskaya | A Fine Size Selection of Brightly Luminescent Water-Soluble Ag-In-S and Ag-In-S/ZnS Quantum Dots[END_REF][START_REF] Podgurska | Effect of Pb 2+ Ions on Photoluminescence of ZnS-Coated AgInS 2 Nanocrystals[END_REF] as well as the injection of ZnS precursors either at room temperature [START_REF] Song | Bandgap and Structure Engineering via Cation Exchange: From Binary Ag 2 S to Ternary AgInS 2 , Quaternary AgZnInS Alloy and AgZnInS/ZnS Core/Shell Fluorescent Nanocrystals for Bioimaging[END_REF][START_REF] Mansur | Eco-Friendly AgInS2/ZnS Quantum Dot Nanohybrids with Tunable Luminescent Properties Modulated by PH-Sensitive Biopolymer for Potential Solar Energy Harvesting Applications[END_REF] or at higher temperature in heat-up, [START_REF] Stroyuk | Origin and Dynamics of Highly Efficient Broadband Photoluminescence of Aqueous Glutathione-Capped Size-Selected Ag-In-S Quantum Dots[END_REF] microwave radiation assisted, [START_REF] Mir | Bandgap Tunable AgInS Based Quantum Dots for High Contrast Cell Imaging with Enhanced Photodynamic and Antifungal Applications[END_REF][START_REF] Mir | Differential and Comparative Sensing Modes of AIS and AIS@ZnS Core-Shell Quantum Dots towards Bioanalytes[END_REF] and hydrothermal [START_REF] Song | Bandgap and Structure Engineering via Cation Exchange: From Binary Ag 2 S to Ternary AgInS 2 , Quaternary AgZnInS Alloy and AgZnInS/ZnS Core/Shell Fluorescent Nanocrystals for Bioimaging[END_REF][START_REF] Song | Facile Synthesis of Water-Soluble Zn-Doped AgIn 5 S 8 /ZnS Core/Shell Fluorescent Nanocrystals and Their Biological Application[END_REF] approaches. In contrast, here the ZnS shell was obtained through the simultaneous, dropwise addition of two distinct precursor solutions, one for zinc and one for sulfur, to the AIS cQDs at elevated temperature (96 °C). It should be noted that the zinc precursor solution contained additional GSH surface ligands to assure the full coverage and colloidal stability of the csQDs. The amount of ZnS shell precursors was calculated to achieve around 3-4 monolayers on the cQDs. Due to the slow injection, the precursor concentration was low enough to prevent separate nucleation of ZnS seeds and to enable the diffusion of the precursor ions to the core particle surface, leading to the formation of a high quality ZnS shell.

From the TEM images in Figure 3a, it was found that the csQDs had an average diameter of 4.8 ± 1 nm, i.e. significantly larger than the core (3.0 ± 0.6 nm). The ZnS thickness is thus estimated as 0.9 nm corresponding to the formation of three monolayers. As mentioned previously, the X-ray diffractogram of the cQDs shows mainly three broad peaks centered at 27.1°, 45.3° and 52.9° that match with the (112), ( 204) and (312) lattice planes of the tetragonal chalcopyrite reference pattern (Fig. 3b). After the shell growth, these diffraction peaks are shifted significantly increased up to a maximum of 55 % for the csQDs (Table 2). The PL line width (full width at half-maximum, FWHM) is about 128 nm for the cQDs and remains almost the same for the csQD (136 nm), while both samples exhibit roughly the same size distribution (around 20%). Thus, it can be concluded that the ZnS shell passivates the surface of the cQDs without introducing further defects. Capping ZnS on cQDs obtained from slow injection was investigated but as expected the maximum PLQY was only 15 % (Fig. S2).

Time-resolved PL analyses (Fig. 4c and4d, Table 1) shows that the decay curve of the csQDs can be very well fitted with bi-exponential model. Hence, the first component (A1, τ1), required for the fitting in the case of the cQDs and corresponding to fast recombination processes on the QD surface defects, does not contribute anymore (Fig. S3, Table S2). The other two decay components are comparable to those of the uncoated QDs. Therefore, the coating of the QD core with the ZnS shell efficiently passivates surface defects responsible in part for PLQY losses through non-radiative transitions.

Summarizing, the developed synthetic approach favors the formation of a thick ZnS shell of three monolayers on the AIS cQDs, leading at the same time to a partial Ag/Zn cation exchange in the core. The obtained Ag0.34Zn0.59InS2/(ZnS)1.69 csQDs exhibit NIR PL emission at around 700 nm with a maximum PLQY of 55 % and a long PL lifetime exceeding 900 ns.

QD conjugation with DNA

As mentioned in the introduction, several ways have been proposed to conjugate QDs with ssDNA. Here, we selected the approach of DNA functionalization during the shell growth because it combines several advantageous features: i) experimentally, the shell growth procedure remains almost the same, just GSH is replaced by thiolated ssDNA; ii) strong conjugation can be expected as the thiolate group of the ssDNA can be integrated into the surface of the ZnS shell (crystal bound rather than surface bound) [START_REF] Turo | Crystal-Bound vs Surface-Bound Thiols on Nanocrystals[END_REF] ; iii) the amount of required functional ssDNA can be minimized. Briefly, the thiolated DNA single strands (molar ratio QD:ssDNAQD= 1:10) were mixed with the Zn precursor solution and then added dropwise together with the S precursor solution (as in the case of standard ZnS shell growth) to the reaction mixture containing the core QDs. The physicochemical parameters used during the synthesis including the salt concentration and the pH have been carefully adjusted to get the highest number of ssDNAQD grafted on the csQDs. According to the optimal conditions reported by Sun and Gang, [START_REF] Sun | DNA-Functionalized Quantum Dots: Fabrication, Structural, and Physicochemical Properties[END_REF] the pH was maintained at 7 and the NaCl concentration did not exceed 0.02 M. We note that the purification of the resulting csQD-DNA from free DNA turned out to be a challenging step, which deserves particular attention. Precipitation as for standard csQDs did not work since free DNA also precipitates upon addition of isopropanol. Another widely used method for the purification of aqueous QDs after functionalization is size exclusion chromatography using gel columns.

However, this method is not suitable neither to remove free DNA molecules due to their high molecular weight (> 30 kDa). On the other hand, gel extraction of conjugated QDs using electrophoresis often results in significant material loss. A few other purification methods reported in literature (ion exchange chromatography using diethylaminoethyl cellulose or ultracentrifugation) are better suited for maintaining the yield and the stability of the bioconjugates, yet their application is tedious. [START_REF] Srinivasan | Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum Dots[END_REF][START_REF] Banerjee | Quantum Dots-DNA Bioconjugates: Synthesis to Applications[END_REF] Therefore, in the present work, the csQD-DNA were purified using centrifugal filters with a 300 kDa molecular weight cut off. The csQD-DNA sample has been carefully purified until no more free DNA strands remain in the solution through absorption measurements (Fig. S4). Finally, the concentrate was redispersed in PBS buffer at pH 7.4.

The obtained csQD-DNA conjugates, which are stable in buffer solution for at least 3 months, exhibit a PL emission maximum of 704 nm and a PLQY of 42 % (Fig. 5a). We attribute the relative reduction of the PLQY by 24 % as compared to csQDs without DNA to the less efficient passivation of the ZnS shell surface induced by the sterically demanding DNA molecules. In the UV-Vis spectra (Fig. 5b), two additional shoulders are observed at around 260 nm and 300 -350 nm in comparison with standard csQDs. From the peak observed at 260 nm, the amount of ssDNAQD on the QD surface can be estimated. The absorption is determined at A260(1mm)= 0.77, hence the concentration of ssDNAQD is 31 µM. The concentration of QDs is estimated to be 5-6 µM, i.e. there are around 5-6 DNA strands per QD (Fig. S5 andS6). This ratio is approximately a factor two lower than that added during the synthesis (QD:ssDNAQD = 1:10), confirming also that unbound ssDNAQD was successfully removed during the purification process.

As an additional technique, agarose gel electrophoresis was used to characterize the DNAconjugated QDs. In fact, this technique, particularly relevant for the separation and purification of biomolecules, is also applied for QDs as a routine technique to reveal the presence of DNA or other biomolecules before and after functionalization. [START_REF] Banerjee | Efficient, and Stable Conjugation of Multiple DNA Strands on Colloidal Quantum Dots[END_REF][START_REF] Zhang | DNA-Based Assembly of Quantum Dots into Dimers and Helices[END_REF][START_REF] Carstairs | DNA Monofunctionalization of Quantum Dots[END_REF][START_REF] Kim | Polyacrylamide Gel Electrophoresis of Semiconductor Quantum Dots and Their Bioconjugates: Materials Characterization and Physical Insights from Spectrofluorimetric Detection[END_REF][START_REF] Hu | Preparation of Biofunctionalized Quantum Dots Using Microfluidic Chips for Bioimaging[END_REF] For comparison, cQDs, csQDs, and csQD-DNA conjugates were measured, taking the former two samples as control. Under an electric field of 120 V, all types of QDs migrated towards the anode, since DNA and GSH surface ligands are negatively charged (Fig. 5c). The electromigration is directly visible and can be monitored in real-time thanks to the luminescence of the QDs through the transilluminator light or through UV light. The mobility is slower for the csQDs than the cQDs while it is faster for csQD-DNA conjugates compared to the standard csQDs. The electrophoretic migration velocity is based on the hydrodynamic size and electric charge of the nanoparticles. For cQDs and csQDs, the difference is caused by the increased overall size of the QDs, while for the csQD-DNA conjugates and csQDs the difference is related to the overall charge on the QD surface: the contribution of DNA charge dominates the electrophoretic particle migration progress in comparison with GSH ligands. The mass predominates the velocity according to the migration of cQDs with regard to csQD-DNA conjugates. Concluding, agarose gel electrophoresis strongly supports the successful conjugation of DNA molecules on the QD surface.

Biological activity of DNA anchored on the QD surface

Although the UV-vis absorption spectra and electrophoresis give strong evidence for the presence of ssDNA on the QDs surface, for further applications of the novel conjugates it is of prime importance to verify that the biological activity of the anchored DNA molecules remained intact. To achieve this, a biochip-based strategy was elaborated using SPRi as detection technique. [START_REF] Anderson | Single Domain Antibody-Quantum Dot Conjugates for Ricin Detection by Both Fluoroimmunoassay and Surface Plasmon Resonance[END_REF][START_REF] Vance | Zeptomole Detection of C-Reactive Protein in Serum by a Nanoparticle Amplified Surface Plasmon Resonance Imaging Aptasensor[END_REF][60] The sensing analysis was carried out on a multiplex biochip with an array of 4 x 6 spots (eight different samples with three replicas) as depicted in Figure 6a and 6b. Half of them included the complementary ssDNASPRcc to that grafted on the QDs (G1, G2, G5, G6) and the others were arbitrary ssDNASPRcontrol (G3, G4, G7, G8) used as negative control, all prepared in four different buffers. Here, several buffers as spotting solutions were used to optimize the conditions for the immobilization and stabilization of these thiolated DNA on the biochip in order to obtain optimal density with sufficient spacing (by electrostatic repulsion) and appropriate orientation for an efficient hybridization afterwards. Furthermore, to minimize nonspecific interactions, the free gold surface on the biochip was blocked with PEG-SH.

Three solutions were injected successively onto the biochip through the SPR micro-fluidic system: csQDs, ssDNAQD and csQD-DNA conjugates. First, the biochip was exposed to two experiments using non-functionalized csQDs (100 nM) for negative control and free ssDNAQD (0.12 µM) for positive control. As illustrated in Figure 6c, there is almost no signal (ΔR< 0.05 %) on all the spots for csQDs injection, showing that there is relatively weak nonspecific adsorption on the biochip. The injection of ssDNAQD (Figure 6d) gave no signal on the spots consisting of the arbitrary ssDNA (negative control). In contrast, it led to significant SPR responses on all the other spots thanks to hybridization with the respective complementary DNA strands. Simultaneously, all these complementary spots were illuminated over the gold surface (Fig. S7). Among these spots, the SPR signal assigned to G5 reached an equilibrium with a maximum of reflectivity variation (ΔR) of nearly 0.8 %. It demonstrated clearly that G5 allowed for the most efficient hybridization. Finally, the solution containing csQD-DNA conjugates at 100 nM was injected. The reflectivity variations increased for all the complementary DNA spots with a particularly high signal for spots obtained with G5 (Fig. 6e), in consistence with results obtained for the injection of ssDNAQD. For better comparison, the sensorgrams in Figure 7b regroup the SPR kinetic curves obtained on the spots of G5 for the three injected samples ssDNAQD (red curve), csQDs (orange curve), csQD-DNA (blue curve) as well as on the spots of G7 for the injection of csQD-DNA (green curve) as negative control. The SPR response is largely enhanced upon the injection of csQD-DNA compared to free ssDNAQD with an amplification factor of 3 (ΔR> 2.5%). Such an enhancement effect on the optical signal was expected since the SPR is a surface sensitive technique. The addition of linked csQD-DNA produced much higher refractive index changes on the SPR surface. In contrast, the negative control spots of arbitrary ssDNA (green curve) did not give reliable response in presence of csQD-DNA. In addition, for illustration, the SPRi differential image obtained after the injection of csQD-DNA was given (cf. The following files are available free of charge.

X-

  Ray diffraction (XRD) measurements were carried out by using a Bruker D8 Advance DaVinci diffractometer with CuKα radiation λ= 1.5406 Å from 2θ= 10-100 °. The chemical composition was determined by energy-dispersive X-ray spectroscopy (EDX) performed on a scanning electron microscope ULTRA 55 (Carl Zeiss AG) with SDD detector (Bruker AXS) and working at 10 kV. Transmission electron microscopy (TEM) analyses were performed on a Technai F20 operating at 200 kV. The samples were deposited onto an ultra-thin graphene oxide layer on a Quantifoil for minimal background from the support film. The UV-Visible absorption spectra were recorded with a NanoDrop 2000c spectrophotometer with an optical path length of 1 mm. The photoluminescence (PL) spectra were obtained from a Horiba Fluorolog-3 model FL3-22 spectrometer (Horiba Jobin Yvon) equipped with 450 W Xe lamp and a R928P detector.

Figure 4 .

 4 Figure 4. The absorption spectra (Figure 4a) do not exhibit any marked features, in accordance

Scheme 1 .

 1 FIGURES

Figure 1 .

 1 Figure 1. TEM images of AgInS2 core QDs obtained from (a)-(d) "slow injection" and from (e)-

Figure 2 .

 2 Figure 2. (a) Schematic representation of AgInS2 core QDs synthesis following either "slow" or

Figure 3 .

 3 Figure 3. (a) TEM images of AgInS2/ZnS QDs with the corresponding size distribution

Figure 4 .

 4 Figure 4. (a) Absorption and (b) PL spectra of the AgInS2/ZnS QDs formation during the shell

Figure 5 .

 5 Figure 5. (a) PL and (b) Absorption spectra of AgInS2 core QDs (black), AgInS2/ZnS core/shell

Figure 6 .

 6 Figure 6. (a) Picture of the biochip array spotted on the SPR prism. (b) Schematic representation

Figure 7 .

 7 Figure 7. (a) Schematic illustration of the hybridization process, (b) Sensorgrams obtained with

Figure S1 :

 S1 Figure S1: PL decay curves of cQDs obtained from fast-and slow-injection fitted with two and

  Conjugation of QDs with biomolecules was first reported by Bruchez et al. and Chan et al. in 1998 employing biotin

Table 1 .

 1 Time-resolved PL data of the core and core/shell samples.

	Sample	χ²	A1 (%) τ1 (ns) A2 (%) τ2 (ns) A3 (%) τ3 (ns)
	Ag1.1InS2.1 cQDs "slow injection"	1.2	1 %	15 ns	13 %	160 ns	86 %	813 ns
	Ag0.53InS2 cQDs "fast injection"	1.2	1 %	26 ns	22 %	317 ns	77 %	994 ns
	Ag0.34Zn0.59InS2/(ZnS)1.69 csQDs	1.3	-	-	24 %	290 ns	76 %	936 ns

Table 2 .

 2 Steady-state PL data of the core and core/shell samples.

	Sample	FWHM (nm)	λem,max	PLQY (%)
	Ag0.53InS2 (core)	128 nm	710 nm	36 %
	Intermediate Shell1	133 nm	707 nm	43 %
	Intermediate Shell2	136 nm	702 nm	50 %
	Ag0.34Zn0.59InS2/(ZnS)1.69	136 nm	696 nm	55 %
	ASSOCIATED CONTENT
	Supporting Information.

ACKNOWLEDGMENT

The authors thank DRF Impulsion for the financial support for the postdoc of Annette Delices.

SyMMES

laboratory is part of Labex Arcane program (ANR-12-LABX-003) and Labex LANEF program (ANR-10-LABX-51-01), both funded by the French National Research Agency. Peter Reiss acknowledges financial support from the French National Research Agency ANR (grants NEUTRINOS, ANR-16-CE09-0015-03 and FLUO, ANR-18-CE09-0039-01). The authors also express their gratitude to Yann Genuist, Daniel Imbert, Stéphanie Pouget and Raphaël Mathey for their fruitful discussions and their contribution related to this project. This work used the platforms of the Grenoble Instruct-ERIC Centre (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The IBS electron microscope facility is supported by the Auvergne-Rhône-Alpes Region, the Fonds FEDER, the Fondation Recherche Médicale (FRM), and the GIS-Infrastructures en Biologie Santé et Agronomie (IBISA). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA)

AUTHOR INFORMATION

Corresponding Authors

K. Kheng -Univ. Grenoble Alpes, CEA, CNRS, IRIG, PHELIQS, F-38000 Grenoble, France; Email: kuntheak.kheng@cea.fr P. Reiss -Univ. Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, UMR5819, F-38000 Grenoble, France; Email: peter.reiss@cea.fr

Notes

The authors declare no competing financial interest.


MD Victoria Lepère

MD Baptiste Duceau

MD, PhD Guillaume Lebreton

MD Camille Bombled

MD Olivier Dujardin

MSc Lucile Boccara

MD Ahmed Charfeddine

MD, PhD Julien Amour

MD, PhD David Hajage

MD, PhD Adrien Bouglé

Risk Factors for Developing Severe Acute Kidney Injury in Adult Patients With Refractory Postcardiotomy Cardiogenic Shock Receiving Venoarterial Extracorporeal Membrane Oxygenation

Keywords: Acute Kidney Injury, Extracorporeal Life Support, Cardiac Surgical Procedure, Cardiopulmonary Bypass, Cardiogenic Shock

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Interventions: None.

Measurements: The primary outcome was the occurrence, within ten days following the VA-ECMO implantation, of a stage 3 AKI defined by the Kidney Disease: Improving Global Outcomes (KDIGO) group.

Main results: One hundred sixty-nine patients (65.7 %) presented with a KDIGO stage 3 AKI; 14 patients (5.4 %) died before the end of the follow-up period, without developing the primary outcome. 92% of patients with KDIGO 3 AKI received RRT, for a median duration of 7 [START_REF] Rastan | Early and late outcomes of 517 consecutive adult patients treated with extracorporeal membrane oxygenation for refractory postcardiotomy cardiogenic shock[END_REF][START_REF] Weisel | Predictors of contemporary coronary artery bypass grafting outcomes[END_REF] days. Late implantation of VA-ECMO was independently associated with an increased risk of KDIGO stage 3 AKI (Odds Ratio 2.81 [95% CI 1.31, 6.07], P = 0.008). The other factors associated with KDIGO stage 3 AKI were pre-operative left ventricular ejection fraction, OR 1.03 [95% CI 1.01, 1.05], P = 0.007; intraoperative plasma transfusion OR 1. [START_REF] Hobson | Cost and Mortality Associated With Postoperative Acute Kidney Injury[END_REF] [95% CI 1.02, 1.26], P = 0.022, increased bilirubinemia level OR 1.013 [95% CI 1.001, 1.026], P = 0.032, and increased creatinine levels OR 1.012 [95% CI 1.006, 1.018], P < 0.001 on the day of implantation.

Conclusions: Significant kidney dysfunction is particularly frequent in patients with refractory PCCS assisted with VA-ECMO. Early implantation of ECMO may help prevent acute kidney injury.

Trial Registration: AKI ECMO Trial Registration: ClinicalTrials.gov Identifier NCT04082312

INTRODUCTION

Cardiogenic shock occurs in 2-6% of patients undergoing cardiac surgery [START_REF] Muehrcke | Extracorporeal membrane oxygenation for postcardiotomy cardiogenic shock[END_REF]. Approximately 1% of patients will require mechanical support using Veno-Arterial ExtraCorporeal Membrane Oxygenation (VA-ECMO) owing to refractory post-cardiotomy cardiogenic shock (PCCS) [START_REF] Smith | An extracorporeal membrane oxygenationbased approach to cardiogenic shock in an older population[END_REF]. Post-cardiotomy VA-ECMO is associated with a high morbidity that includes bleeding, ischemic events, and organ failure including acute kidney injury (AKI) [START_REF] Rastan | Early and late outcomes of 517 consecutive adult patients treated with extracorporeal membrane oxygenation for refractory postcardiotomy cardiogenic shock[END_REF]. Indeed, AKI is one of the most frequent complications in patients supported by ECMO, with an incidence reaching up to 65% [START_REF] Haneya | Impact of Acute Kidney Injury on Outcome in Patients With Severe Acute Respiratory Failure Receiving Extracorporeal Membrane Oxygenation[END_REF]. Renal replacement therapy (RRT) is required in approximately 65% of patients [START_REF] Rastan | Early and late outcomes of 517 consecutive adult patients treated with extracorporeal membrane oxygenation for refractory postcardiotomy cardiogenic shock[END_REF], and is associated with increased mortality [START_REF] Ko | Extracorporeal membrane oxygenation support for adult postcardiotomy cardiogenic shock[END_REF]. Through these protective mechanisms the timing of VA-ECMO implantation may influence the prognosis of patients with refractory PCCS. Our hypothesis was that early implantation of VA-ECMO may decrease the incidence of AKI in patients with PCCS. The aim of our study was to describe the incidence of acute kidney injury in adult patients with refractory PCCS supported by VA-ECMO and identify the risk factors for developing AKI.

METHODS

This study was approved by the ethics committee Ile de France 5 (reference B-7-15). Due to the retrospective design of the study, and in accordance with the decision of the ethics committee and French regulation and non-interventional studies, signed informed consent was waived. The study was conducted in accordance with the Declaration of Helsinki principles.

Design and setting

This observational single center retrospective study was conducted in the Surgical Intensive Care Unit of the Cardiology Institute of La Pitié-Salpêtrière University Hospital (Paris, France). All patients who underwent cardiac surgery admitted in our unit and assisted by VA-ECMO between January 2013 and December 2016 were screened. The exclusion criteria were i) chronic hemodialysis, ii) death occurring within the first 48 hours of VA-ECMO implantation and iii) multiple VA-ECMO episodes during the same hospitalization. Patients with VA-ECMO were identified through billing record in the electronic medical records system. All patient data was anonymized.

Main goals and Outcomes

Our primary objective was to describe the incidence of acute kidney injury in adult patients with refractory PCCS supported by VA-ECMO and identify the risk factors for developing AKI. The primary outcome was the occurrence of a stage 3 AKI defined by the Kidney Disease: Improving Global Outcomes (KDIGO) group (Supplemental Appendix) (6) within ten days of VA ECMO implantation. Stage 3 AKI was selected due to its potential for requiring RRT. During this ten days period (D1 to D10), AKI and death behave as competitive events (D1 represents the day following the implantation of ECMO). The primary outcome group was divided as follows (i) patients with stage 3 AKI within 10 days of VA ECMO implantation (group 1); (ii) patients without stage 3 AKI 10 days after VA ECMO implantation (group 2, baseline category); (iii) patients that died between D2 and D10 of VA-ECMO support, without developing KDIGO stage 3 AKI (group 3). Death occurring before D2 of VA-ECMO implantation was an exclusion criterion.

Our second goal was to determine the short-term mortality risk factors in this population. The secondary outcome was defined as 30-day mortality after VA-ECMO implantation. Longterm outcomes including the 6-month mortality rate, and the 1-month and 6-month renal prognosis were also assessed.

Statistical analysis

Results are expressed as number of patients (%), median (interquartile range 25-75) or mean (± standard deviation). Shapiro-Wilk test was used to assess the normality assumption for continuous variables. The groups were compared using Kruskal-Wallis, Pearson Chi-square and Fisher tests. Univariable analyses and multivariable regression models were used to identify the risk factors for developing the primary and the secondary outcomes. Risk factors for the 30-day mortality were demographic factors (age, BMI, SAPS 2, SOFA, LVEF, diabetes, dyslipidemia, hypertension, peripheral artery disease), intraoperative factors (transfusion), variables at VA-ECMO implantation (biventricular failure, primary graft dysfunction, lactate, inhaled nitric oxide, AKI at implantation, urine output), and type of surgery (heart transplantation, valve surgery, coronary bypass surgery, combined surgery).

Regarding the secondary outcome, the final logistic regression model was obtained using the same selection method as used for the analysis of the primary outcome.

Discrimination abilities was quantified using the Polytomous Discrimination Index (PD-Index) [START_REF] Van Calster | Assessing the discriminative ability of risk models for more than two outcome categories[END_REF] for the final multinomial model of the occurrence of stage 3 AKI, and the Concordance Index (C-Index) [START_REF] Van Calster | Assessing the discriminative ability of risk models for more than two outcome categories[END_REF] for the binary logistic model of 30-day mortality. Internal validation was performed with a bootstrap procedure using 500 repetitions [START_REF] Steyerberg | Prognostic modelling with logistic regression analysis: a comparison of selection and estimation methods in small data sets[END_REF], to quantify the optimism that may be expected when the multivariable model is applied to new, but similar, patients. Both apparent and optimism-corrected discrimination indexes were reported Odds Ratio (OR) with their 95% confidence intervals (CI) was calculated. The complete-case method was used for the multivariable analyses. A sensitivity analysis using multiple imputations replaces missing values when appropriate was also performed (Supplemental Table 2). All tests were 2-sided and a P-value < 0.05 was considered for statistical significance. Statistical analyses were performed using R software (version 3.4.1, licenses GNU GPL, The R foundation for statistical computing, Vienna, Austria).

RESULTS

Study population

Between January 2013 and December 2016, 4796 patients underwent cardiac surgery with cardiopulmonary bypass. Among them, 347 (7.2%) experienced refractory PCCS and required VA-ECMO assistance (peripheral cannulation 94.8%) (Figure 1, flowchart). 257 patients were included in the final analysis. Demographic and outcome variables did not significantly differ between the patients included and excluded from the analysis. One hundred sixty-nine (65.7%) patients developed KDIGO stage 3 AKI within 10 days of VA-ECMO support. Demographic and clinical characteristics of the population are described in Table 1. The clinical management of VA-ECMO patients is described in the Supplementary Appendix.

VA-ECMO implantation

Risk factors for stage 3 AKI

During the follow-up period of ten days after VA-ECMO support, 169 patients (65.7 %) developed a KDIGO stage 3 AKI; 74 patients (28.8 %) did not develop a KDIGO stage 3 AKI and were still alive at D10; 14 patients (5.4 %) died before the end of the follow-up period, without developing the primary outcome. Renal replacement therapy was initiated in 156 patients (60.7%) from the overall cohort. 92% of patients with KDIGO stage 3 AKI received RRT for indications including ionic disorders (No.=79, 46.7%), anuria (No.=61, 36.1%) and fluid overload (No.=15, 8.9%). The median duration of RRT was 7 [IQR 3, 16] days.

Results from the univariate analysis assessing risk factors for developing KDIGO stage 3 AKI are presented in Table 2. SOFA, SAPS2, and urine output at Day 1 were excluded from the analysis due to a lack of data (>15% of data points missing). Figure 2 summarizes the results of the multivariate logistic regression. Factors independently associated with the development of KDIGO stage 3 AKI were bilirubinemia and creatinine levels on the day of ECMO implantation, the timing of ECMO implantation, pre-operative LVEF and intraoperative fresh frozen plasma transfusion. Complete multivariate results including group 3 patients is available in the Supplemental Table 1. The Polytomous Discrimination Index (PD-Index) for the final multivariate model of the occurrence of stage 3 AKI was 0.624. After internal validation, the optimism-corrected discrimination index was 0.294.

Risk factors for 30-day mortality

The 30 day mortality rate was 48.2% (124/257). The timing of VA-ECMO implantation did not reduce the mortality at D30 in the univariate analysis, with an intraoperative mortality rate of 44.7% (No.=71/159) vs. 54.1% (No.=53/98) for postoperative implantation OR 0.69 [0.40, 1.17] P=0. [START_REF] Weisel | Predictors of contemporary coronary artery bypass grafting outcomes[END_REF]. The 6 month mortality rate was 75.9% (n=154/203). Intraoperative implantation of VA-ECMO was associated with a lower mortality at 6 months, 69.8% (No. =88/126, 20.8% missing status) compared to 85.7% (n=66/77, 21.4% missing status) for patients with postoperative implantation, OR=0.39 [0.17, 0.85], P=0.011. The factors associated with 30-day mortality after VA-ECMO implantation are presented in the Table 3.

The Concordance Index (C-Index) for the binary logistic model of 30-day mortality was 0.721. After internal validation, the optimism-corrected discrimination index was 0.482.

Renal prognosis at one month (Supplemental Table 3) was good, with all but three patients recovered from their stage 3 AKI at 6 months.

DISCUSSION

To our knowledge, this study is the first to evaluate the risk factors for developing AKI in adult patients with refractory PCCS. In this population, intra-operative implantation of VA-ECMO appeared to be a protective factor for developing a stage 3 AKI.

The incidence of KDIGO stage 3 AKI in our cohort was 65.7%. This is higher than prior reported values from postoperative cardiac surgical patients without VA-ECMO assistance ( 9), but consistent with the results from Yan et al., who found an incidence of severe AKI of 48% at 48 hours after VA-ECMO implantation [START_REF] Yan | Acute kidney injury in adult postcardiotomy patients with extracorporeal membrane oxygenation: evaluation of the RIFLE classification and the Acute Kidney Injury Network criteria[END_REF]. The use of renal replacement therapy was higher than previously described, with initiation of RRT in 60.7% of patients in the overall cohort [START_REF] Elella | Incidence and Outcome of Acute Kidney Injury by the pRIFLE Criteria for Children Receiving Extracorporeal Membrane Oxygenation after Heart Surgery[END_REF]. Typically, patients needing VA-ECMO support have a higher comorbidity index and lower pre-operative LVEF. These patients are thus at higher risk for postoperative complications, including AKI. Additionally, the relatively high incidence of refractory PCCS in our cohort reflects the underlying health status of the population of an expert university hospital, with mean EuroSCORE II [START_REF] Nashef | EuroSCORE II[END_REF] predicting the intraoperative mortality of almost 20%.

The results of this study magnify the importance of the timing around implantation of VA-ECMO support. We found that intraoperative implantation of VA-ECMO had a protective effect on the development of KDIGO stage 3 AKI. Intraoperative implantation of VA-ECMO limits the duration of the low cardiac output associated with PCCS and thus helps prevent subsequent organ dysfunction. Hence, biological marker of organ dysfunction as hyperbilirubinemia was independent risk factor of KDIGO stage 3 AKI in our cohort.

Due to the design of our study, we were not able to assess the effect of developing an AKI 3 between D2-D10 on the 30 day mortality rate. The overlap between the exposition period to AKI and the mortality risk prevented modeling of the relationship between these two variables. Nonetheless this association has been extensively documented in past studies [START_REF] Hobson | Cost and Mortality Associated With Postoperative Acute Kidney Injury[END_REF][START_REF] Mao | Cardiac surgery-associated acute kidney injury[END_REF][START_REF] Shah | Clinical outcomes of 84 children with congenital heart disease managed with extracorporeal membrane oxygenation after cardiac surgery[END_REF]. Given the high incidence of AKI, with 89% of patients developing at least KDIGO stage 1 AKI, preventative strategies may have a major impact on patient outcomes. The other 30day mortality risk factors highlighted by our study are commonly described in both a postcardiotomy population [START_REF] Weisel | Predictors of contemporary coronary artery bypass grafting outcomes[END_REF] and in VA-ECMO population [( 15);( 17)], including age, BMI, the presence of chronic arteriopathy and biventricular failure. The supplemental figure (Diagram Acyclic Graph) summarizes the results and the hypotheses stemming from this study and provides additional context with respect to cardiac surgery-associated AKI.

Our study has several limitations. First, because of its retrospective and single center design, unidentified confounding factors may have biased the multivariable analyses. Nonetheless, the risk factors we identified are consistent with the ones previously described, providing us with some external validity. Second, if missing data did not allow a full-set analysis, we used a sensitivity analysis using multiple imputations to replaces missing values. Third, the lack of long term follow-up, inherent to the retrospective design, prevented us from making conclusions regarding the effect of long-term mortality or renal outcome. Fourth, there was no detailed protocol regarding cardiopulmonary bypass weaning, decision for VA-ECMO implantation in case of refractory PCCS, or RRT initiation in the intensive care unit. However, our practices are relatively standardized, and it is unlikely that a patient with refractory PCCS during the perioperative period would not have been assisted by VA-ECMO.

CONCLUSIONS

In conclusion, this retrospective study is the first to emphasize the role of intraoperative VA-ECMO support in case of refractory PCCS, which could possibly decrease the incidence of KDIGO stage 3 AKI. AKI is the easier read-out of low cardiac output for intensivist, and other organ dysfunction might be avoided with early VA-ECMO support. Only a prospective study with randomization could definitely clarify the issue of timing in VA-ECMO support during PCCS. Refractory Post-Cardiotomy Cardiogenic Shock n=354

  In the case of PCCS, two perioperative mechanisms may play a role in the development of AKI. First, low cardiac output associated with left ventricular failure leads to a decline in renal blood flow and glomerular filtration. Second, right ventricular failure may also impair glomerular filtration due to renal congestion. Both renal congestion and low cardiac output are treated by VA-ECMO which helps increase flow rate and arterial pressure while decreasing central venous pressure.

  Risk factors analyzed included demographic factors (SAPS 2, SOFA, Pre-operative LVEF, chronic kidney failure, creatinine clearance, type of surgery), hemodynamic factors during intraoperative period (cardiopulmonary bypass time, transfusion, catecholamines), factors associated with VA-ECMO implantation (primary graft dysfunction, lactate, intraoperative or postoperative implantation, catecholamines), and biological factors at D1 of VA-ECMO support (creatinine, blood protein level, bilirubinemia, bicarbonates, lactate, urine output). A multivariate logistic regression was used to model the occurrence of stage 3 AKI. This model was chosen to take into account the competing risk of death without developing a stage 3 AKI. The three levels consisted of patients with KDIGO stage 3 AKI (group 1), patients without KDIGO stage 3 AKI and alive at D10 (group 2 [baseline category]), and patients that died before D10 without developing KDIGO stage 3 AKI (group 3). Candidate risk factors associated (p < 0.05) with this outcome in the univariable analysis were introduced into the multivariate model, and the final model was selected using backward stepwise selection method.

Figure 1 .

 1 Figure 1. Flowchart

Figure 2 .

 2 Figure 2. Adjusted odds ratios of KDIGO stage 3 Acute Kidney Injury.

Table 2

 2 shows data regarding perioperative management of the patients. The VA-ECMO was implanted intraoperatively in 159 patients (61.9%) and postoperatively in 98 patients (38.1%).

	A left ventricular venting was necessary in 105 patients (40.9%), most commonly an intra-
	aortic balloon pump per institutional protocol (No.=92, 35.8%). The causes of VA-ECMO
	implantation were left ventricular failure (No.=76, 29.6%), right ventricular failure (No.=45,
	17.5%), biventricular failure (No.=88, 34.2%), primary graft failure (No.=30, 11.7%) or
	cardiac arrest (No.=15, 5.8%).

Table 1 :

 1 Multivariable analysis of risk factors for AKI in patients going under cardiac surgery supported by VA-ECMO

	Odds Ratio [95% CI]	P-value
	1.03 [1.01, 1.05]	0.007
	1.012 [1.006, 1.018]	<0.001
	1.013 [1.001, 1.026]	0.032
	2.81 [1.31, 6.07]	0.008
	1.13 [1.02, 1.26]	0.022

Table 2 :

 2 Univariable analysis of potential risk factors for KDIGO stage 3 AKI

		Patients with	Patients without stage 3 AKI
		stage 3 AKI	alive	deceased
			at D10	before D10
	Factors	(No.=169)	(No.=74)	(No.=14)	P-value
	Demographic characteristics and comorbidities
	SAPS 2	61.1 ± 18.3	53.7 ± 16.7	56.4 ± 19.2	0.043
	SOFA	16.8 ± 3.2	11.7 ± 2.9	12.6 ± 3.9	<0.0001
	Preoperative LVEF (%)	37.3 ± 16.9	30.6 ± 16.7	36.9 ± 16.6	0.011
	Chronic kidney failure a	46 (27.2)	21 (28.4)	2 (14.2)	0.61
	Creatinine clearance, ml/min	68 [46, 89]	73 [55, 97]		0.15
	Type of surgery				0.0503
	Heart transplant	29 (17.2)	28 (37.8)	2 (14.3)
	Valvular surgery	51 (30.2)	15 (20.3)	4 (28.6)
	Coronary bypass	36 (21.3)	11 (14.8)	2 (19.0)
	Combined surgery	17 (10.0)	4 (5.4)	1 (7.1)
	Others	36 (21.3)	16 (21.6)	5 (35.7)
	Intraoperative period
	Cardiopulmonary Bypass Time, min	123 [87, 181]	123 [89, 166]	123 [78, 158]	0.85
	Red Blood Cell transfusion, number	2 [0, 5]	2 [0, 4]	3 [2, 6.5]	0.37
	Platelet transfusion, number	1 [0, 1]	1 [0, 1]	1 [0.25, 1.75]	0.68
	Plasma transfusion, number	3 [0, 6]	3.5 [0, 5]	6.5 [3, 8.75]	0.047
	Cell salvage autologous transfusion, mL	450 [230, 682]	423 [240, 533]	717 [315, 840]	0.27
	Epinephrine, yes/no	97 (57.4)	39 (52.7)	10 (71.4)	0.42
	Epinephrine, µg/kg/min b	0.10 [0, 0.31]	0.07 [0, 0.22]	0.15 [0.02, 0.45]	0.17
	Dobutamine, yes/no	50 (29.6)	31 (41.9)	5 (35.7)	0.17
	Dobutamine, µg/kg/min b	0 [0, 5]	0 [0, 7]	0 [0, 9.5]	0.32
	Norepinephrine, yes/no	87 (51.5)	33 (44.6)	7 (50)	0.61
	Norpinephrine, µg/kg/min b	0.02 [0, 0.19]	0 [0, 0.19]	0.18 [0, 0.45]	0.36
	VA-ECMO implantation
	Primary graft dysfunction	12 (7.1)	16 (21.6)	2 (14.3)	0.006
	Lactate, mmol/L b	6.9 (3.8)	5.1 (3.2)	5.2 (2.7)	0.002
	Intraoperative implantation	90 (53.2)	57 (77.0)	12 (85.7)	0.0004
	Postoperative implantation	79 (46.7)	17 (22.9)	2 (14.3)	0.0004
	Epinephrine, yes/no	123 (72.8)	42 (56.8)	10 (71.4)	0.049
	Dobutamine, yes/no	36 (21.3)	27 (36.5)	4 (28.6)	0.046

Table 3 :

 3 Risk factors for D30 mortalityResults are count (%), mean ±SD or median [interquartile range 25, 75]. Adjusted Odds Ratio of factors that did not achieve statistical significance in multivariable analysis are not presented.Due to missing data, SOFA and SAPS2 were not included in the multivariable analysis. AKI, Acute Kidney Injury; BMI, body mass index; COPD, chronic obstructive pulmonary disease; LVEF, left ventricular ejection fraction; SAPS 2, simplified acute physiology score 2; SOFA, Sepsis-related Organ Failure Assessment; VA-ECMO, Veno-Arterial ExtraCorporeal Membrane Oxygenation.

				Univariable	Multivariable
		Deceased	Alive	analysis	analysis
		at D30	at D30		Odds Ratio	P-value
	Factors	No.=124	No.=133	P-value	[95%CI]
	Demographic characteristics
	Age, years	66.8±11.8	60.3±13.5	<0.001	1.007 [1.002,	0.003
					1.012]
	BMI, kg/m 2	26.8±5.7	24.5±4.3	<0.001	1.015 [1.003,	0.013
					1.026]
	SAPS 2	63.7±18.4	48.4±16.3	<0.001	-	-
	SOFA	16.5±3.7	13.6±3.5	<0.001	-	-
	LVEF, %	40 [25, 50]	25 [20, 45]	<0.001	-	-
	Diabetes	40 (32.3%)	28 (21.1%)	0.058
	Dyslipidemia	77 (62.1%)	60 (45.1%)	0.009	-	-
	High blood pressure	81 (65.3%)	59 (44.4%)	0.001	-	-
	Chronic arteriopathy	24 (19.4%)	9 (6.8%)	0.005	1.20 [1.01-1.44]	0.041
	Intraoperative period
	Red Blood Cell	3 [0, 5]	2 [0, 4]	0.091	-	-
	transfusion, number
	Cell salvage autologous	475 [247, 725]	392 [228, 663]	0.082	-	-
	transfusion, mL
	VA-ECMO implantation
	Biventricular failure	53 (42.7%)	35 (26.3%)	0.008	1.15 [1.02-1.30]	0.028
	Primary graft dysfunction	22 (16.5%)	8 (6.5%)	0.020	-	-
	Lactate, mmol/L	6.1 [3.4, 9.2]	5.3 [3.0, 8.4]	0.069	-	-
	Inhaled Nitric Oxid	10 (8.1%)	24 (18.0%)	0.030	-	-
	AKI at implantation	76 (61.3%)	63 (47.4%)	0.035	-	-
	Urine output	750 [200, 1390]	1125 [500, 1860]	0.008	-	-
	Type of surgery			0.003	-	-
	Heart transplantation	15 (12.1%)	44 (33.1%)	-	-	-
	Valvular surgery	38 (30.6%)	32 (24.1%)	-	-	-
	Coronary bypass	27 (21.8%)	22 (16.5%)	-	-	-
	Combined surgery	13 (10.5%)	9 (6.8%)	-	-	-
	Other surgery	31 (25.0%)	26 (19.5%)	-	-	-

ACT, Activated whole blood Clotting Time AKI, Acute Kidney Injury ASA, American Society of Anesthesiologists BMI, Body Mass Index CI, Confidence Intervals COPD, Chronic Obstructive Pulmonary Disease DO2, Dissolved O2 ECMO, ExtraCorporeal Membrane Oxygenation ELSO, Extracorporeal Life Support Organization KDIGO, Kidney Disease: Improving Global Outcomes LVEF, Left Ventricular Ejection Fraction OR, Odds Ratio PCCS, Post-Cardiotomy Cardiogenic Shock pCO2, CO2 Partial Pressure pO2, O2 Partial Pressure RRT, Renal Replacement Therapy SAPS 2, simplified acute physiology score 2 SOFA, Sepsis-related Organ Failure Assessment VA-ECMO, Veno-Arterial ExtraCorporeal Membrane Oxygenation

ACKNOWLEDGMENTS

None.

Conflict of interest

The authors have not disclosed any potential conflict of interest regarding this study.