Method for selective surface modification of an object

Abstract

A process for selective surface modification of an object comprising at least one SiN surface and at least one silicon oxide (SiO) surface, the process comprising the steps of a. Treating the object with hydrogen fluoride (HF) solution to obtain an etched object; b. Treating the etched object with a polymer solution comprising a polymer having a net negative charge to obtain an object having a polymer coating on the at least one SiN surface; c. Optionally irradiating the object having a polymer coating on the at least one SiN surface with ultraviolet light to obtain an object having a crosslinked polymer coating on the at least one SiN surface; d. Optionally treating the object having a (optionally crosslinked) polymer coating on the at least one SiN surface with a silane to obtain an object with a silane coating on the at least one SiO surface.

Description

[0001] METHOD FOR SELECTIVE SURFACE MODIFICATION OF AN OBJECT

[0002] Field

[0003] The invention relates to a process for selective surface modification of an object comprising at least one silicon nitride (SiN) surface and at least one silicon oxide (SiO) surface, a sensor chip obtainable by the process, and a biosensor comprising a sensor chip according to the invention.

[0004] Background

[0005] Silicon nitride (SiN) and silicon oxide (SiO) are widely used materials for microfabrication of devices, such as electronic or photonic integrated circuits. SiN may for example be used as a dielectric insulator, barrier, and passivation layer, or as a planar optical waveguide. SiO may for example be used as a dielectric passivation layer, as gate dielectric in field-effect transistors, or as the cladding of SiN planar optical waveguides.

[0006] Many devices comprise both SiN and SiO surfaces. In such devices, the position and structure of the different used materials is carefully considered in view of their function. Besides the bulk properties, also the surface properties of materials play an important role in the functioning of devices. The desired surface properties of different materials may therefore also be different.

[0007] For example, in a photonic biosensor device comprising a SiN planar optical waveguide embedded in a SiO cladding, wherein the cladding has been partly removed to expose the surface of the SiN waveguide to the sample solution, the SiN surface needs to be modified to immobilize bioreceptors which can recognize and bind the analyte of interest. On the other hand, adsorption of biomolecules on the SiO surface (biofouling) has to be prevented as much as possible. Thus, the desired properties of the SiN and the SiO surfaces are different, which requires a selective surface modification method.

[0008] Several methods for the surface modification of SiO and SiN are known. The most widely used method is the application of self-assembled monolayers (SAMs) of hydrolysable silanes, e.g. chloro- or alkoxysilanes. These silanes react with surface silanol groups in a condensation reaction, resulting in the formation of a siloxane bond (Sisurface-O-Sisiiane) and release of H2O. However, silanol groups are generally present on both SiO and SiN surfaces in ambient conditions. Therefore, treatment of an object, comprising at least a SiO and a SiN surface, with a hydrolysable silane will result in uniform modification of the surface having SiO and SiN. Thus, the surface properties of the SiN and the SiO surface will be the same, while this may not be preferred in view of their function in the device. Thus, there is a need for advanced surface modification methods which can provide selective surface modification of SiN and / or SiO.

[0009] Other known methods for the surface modification of SiN comprise the step of treating the surface with HF to remove the native oxynitride. It has been shown that HF treated SiN surfaces mainly comprise Si-F groups (approximately 70%), and smaller amounts of Si-OH and Si-NH2 groups [liu - j phys condens matter 28 (2016) 094014], US8221879 discloses surface modification of SiN with alkenes / alkynes. The SiN surface is treated with e.g. HF to remove native oxynitride and subsequently reacted with alkenes / alkynes to form a molecular monolayer on SiN. The reaction can be performed for 2-24 hours at high temperature, e.g. 200 °C.

[0010] US8481435 discloses a similar process wherein the alkenes / alkynes are reacted photochemically for 16 hours under UV irradiation in oxygen- and water-free conditions. The photochemical method has been applied to objects comprising both SiN and SiO surfaces, and it has been shown that this results in material-selective modification of the SiN surface [knoben - proc microTAS 22 (2018) 982], However, this method is very unpractical, since the reaction has to be performed for a very long time (for example at least 16 hours) under UV irradiation and in water- and oxygen-free conditions. Also, scale up of said method is difficult.

[0011] Material-selective surface modification of SiN vs. SiO can also be achieved by reaction of amine groups on the HF etched SiN surface with aldehydes. This reaction takes 2 hours at room temperature, and requires the use of deoxygenated solutions and an argon atmosphere [bahuls - biosens bioelectron 25 (2010) 1460], or 16 hours at 65 °C under nitrogen atmosphere [liu - j phys condens matter 28 (2016) 094014], The use of inert atmosphere and long reaction times make also this reaction of limited practical use. Furthermore, the effectiveness of this method is limited since it is based on reaction of the small amount of Si-NH2 groups present on the HF treated SiN surface. It has been shown that amine groups on the SiN surface can also be reacted with azides in 15 minutes under UV irradiation [hoi - biosens bioelectron 102 (2018) 497], However, this method involves an activation process of the SiN surface using hydrogen plasma generated by ICP-RIE (Inductively Coupled Plasma - Reactive Ion Etching), which plasma is difficult to use. This makes this method complex to implement. Furthermore, the effectiveness of this method is limited since it is based on reaction of the small amount of Si-NH2 groups present on the HF treated SiN surface.

[0012] Another example discloses treatment of SiN by (buffered) HF, followed by different methods for generating azide groups on the surface, which are then reacted with alkynes using Cu click (CuAAC) chemistry [dietrich - appl surf sci 481 (2019) 10], It is not mentioned if this method results in selective modification of SiN vs. SiO. Although the CuAAC reaction takes place under mild conditions (aqueous solution, room temperature), the reaction is performed for 24 hours in the dark. The long reaction time and special conditions limit the practical usefulness of the method. Furthermore, the effectiveness of this method is limited since it is based on reaction of the small amount of Si-NH2 groups present on the HF treated SiN surface.

[0013] KANG MC et al , Japanese Journal of Applied Physic, Vol.44, No.8, 2005, pp 5949-5952 describe the effect of organic additives on ceria slurry in shallow trench isolation chemical mechanical planarization. The aim was to increase the removal selectivity between silicon oxide and silicon nitride. When polyacrylic acid was used in a ceria slurry, the polyacrylic acid served as protector of the silicon nitride due to change in zeta potential.

[0014] Kenji Mochida et al, Microelectronic Engineering, 276, p.112001 (2023) disclose a simple method of selectively functionalizing Si3N4 with a protective polymer film to reduce the etching rate of Si3N4 relative to the etching rate of SiO2. A water-soluble cellulose based polymer, DC01 , was found to reduce the etching of Si3N4 selectively.

[0015] Chinese patent application CN116169562A relates to colloidal quantum dot light source integrated structures and preparation methods thereof. Figure 1 discloses a structure comprising a dense colloidal quantum dot layer(05) layer that may be applied by a spin-coating process and organic crosslinking material. Said layer is deposited on top of a silicon nitride lower cladding layer (04). On top of the dense colloidal quantum dot layer, a silicon nitride cladding layer (06) is present.

[0016] Heideman et al, in Proc, of SPIE Vol. 11238 112830Q-1 , disclose ultrasensitive photonic integrated circuit based biosensors for healthcare applications. The paper describes in some detail the chips used in the biosensor.

[0017] Problem to be solved

[0018] The problem to be solved is to provide a process for the surface modification of an object comprising both a SiN and a SiO surface, which selectively modifies the SiN surface, and leaves the SiO surface substantially unmodified. In a preferred embodiment the SiN and SiO surfaces are independently modified in a different way. In particular, it is an object of the present invention to provide a process for the selective modification of the SiN surface of an object comprising both a SiN and a SiO surface. It is also an object to provide a process that requires fewer steps and takes less time, and that can be carried out under milder conditions compared to the state of the art. It is also an object to provide a more effective surface modification process, i.e. a process that results in a modified SiN surface with a higher density of functional groups. It is further an object to prepare an object having selectively modified SiN and SiO surfaces for use in a (bio)sensor, in order to improve the selectivity and / or sensitivity of the (bio)sensor.

[0019] Summary of the invention

[0020] The present invention provides a process for selective surface modification of an object comprising at least one silicon nitride (SiN) surface and at least one silicon oxide (SiO) surface, the process comprising the steps of a. Treating the object with hydrogen fluoride (HF) solution to obtain an etched object; b. Treating the etched object with a polymer solution comprising a polymer having a net negative charge to obtain an object having a polymer coating on the at least one SiN surface, while the SiO surface is substantially unmodified; c. Optionally irradiating the object having a polymer coating on the at least one SiN surface with ultraviolet light to obtain an object having a crosslinked polymer coating on the at least one SiN surface; d. Optionally treating the object having a (crosslinked) polymer coating on the at least one SiN surface with a silane to obtain an object with a silane coating on the at least one SiO surface.

[0021] Preferably, in the process according to the invention, prior to step a) the object has undergone an activation treatment. It is an advantage of the process wherein prior to step a) the object has undergone an activation treatment that the polymer coating formed in step b) is obtained in a more reproducible manner. Ultimately, more reproducible polymer coatings make it possible to provide sensor chips with a better reproducibility.

[0022] In the framework of the invention, a SiN surface is a surface of preferably SisNy, wherein y ranges from 1 to 4, preferably from 2.5 to 4. The SiN surface most preferably is a surface of SiaN^ Typically, some oxidation of the SiN surface occurs in ambient conditions, resulting in the presence of some oxynitride. In some cases, some contaminants are also present on the SiN surface.

[0023] In the framework of the invention, an SiO surface is a surface of preferably SiOx, wherein x ranges from 1 to 2, preferably from 1.5 to 2. The SiO surface most preferably is a surface of SiC>2. In some cases, some contaminants are also present on the SiO surface.

[0024] In the framework of the invention, a surface is considered to be selectively modified if the thickness of the surface modification layer on SiN is greater than or equal to 0.5 nm, and the thickness of the surface modification layer on SiO is less than or equal to 0.1 nm, whereby the thickness of the surface modification layers is measured by XPS as described below.

[0025] Preferably, the thickness of the surface modification layer on SiN is at least 0.8 nm and the thickness of the surface modification on SiO is equal to or less than 0.1 nm. More preferably, the layer thickness of the surface modification layer on SiN is at least 1 nm and the thickness of the surface modification layer on SiO is equal to or less than 0.1 nm, most preferably less than 0.1 nm.

[0026] In contrast to the other methods described above, the process according to the invention is not based on reaction of a specific group (such as Si-NH2 or Si-H) of which only a small amount is present on the surface, but on physical interactions between the negatively charged polymer and the SiN surface. It is therefore an advantage of the process that it allows more effective surface modification of SiN and results in SiN surfaces which are coated and have a higher density of functional groups. It is a further advantage of the process according to the invention that it allows the production of sensor chips with a good sensitivity / selectivity for analytes of interest.

[0027] In the process according to the invention, an object is treated with a HF solution to obtain an etched object. HF treatment is used to provide a clean SiN surface for subsequent polymer adsorption. It is known that HF quickly etches SiO and oxynitrides, but not SiN. For example, the etch rate of annealed SiO2 in 1 wt% HF is approximately 4 nm / min. Therefore, the HF treatment time should be as short as possible to minimize etching and damage of SiO, but long enough to effectively remove oxynitride and organic contaminations from the SiN surface. Typically, the HF solution is an aqueous solution. The concentration of HF in the HF solution may vary, and may lie for example between 0.5 and 3 wt% HF, relative to the weight of the HF solution. Typically, a 1 wt% solution of HF in water is used.

[0028] Typically, the object is treated with the HF-solution by submerging it in the solution. Preferably the treatment time ranges between 20 seconds and 5 minutes, at a temperature between 15 and 30 °C, preferably 18-25 °C. For a 1 wt% HF solution in water, a suitable time for keeping an object with an SiN / SiO surface submersed is e.g. between 60 and 120 seconds, preferably between 80 and 100 seconds, at a temperature between 18-25 °C.

[0029] In the framework of this invention, a surface is considered to be clean if after the treatment with HF no carbon is detected anymore on the surface with XPS.

[0030] In the process according to the invention, prior to step a), the object preferably has undergone an activation treatment. The aim of the activation treatment is to remove organic contaminants that may be present on the surface of the object. The activation treatment may for example comprise an oxidation treatment, for example exposure to air plasma or oxygen plasma, to a piranha solution, or UV / ozone treatment. Preferably, the oxidation treatment comprises exposure to air plasma or oxygen plasma. It is an advantage of the the process according to the invention wherein prior to step a) the surface has undergone an activation treatment that the polymer coating formed in step b) is obtained in a more reproducible manner. Ultimately, more reproducible polymer coating makes it possible to provide sensor chips with a better reproducibility.

[0031] In the process according to the invention, the etched object obtained after step a) is treated with a polymer solution comprising a polymer having a net negative charge to obtain an object having a polymer coating on the at least one SiN surface.

[0032] In the framework of the invention, the polymer having a net negative charge has a negative charge when present in the solution used to treat the etched object. Preferably the negative charge is present in pendant groups of the polymer.

[0033] In the framework of the invention, a pendant group is defined as a group of atoms attached to the backbone chain of a polymer. Preferably at least part of the pendant groups are negatively charged. In the framework of the invention, the pendant groups preferably have a negative charge when present in the polymer solution.

[0034] Preferably the polymer comprises at least 1 chargeable pendant group per 10 carbon atoms of the backbone of the polymer, in order to provide a sufficiently negatively charged polymer in solution. More preferably, the polymer comprises at least 1 chargeable pendant group per 5 carbon atoms of the backbone of the polymer. Most preferably, the polymer comprises less than or equal to 1 chargeable group per 2 carbon atoms of the backbone of the polymer.

[0035] Suitable groups that may provide a negative charge to the polymer in solution are for example a carboxylate group, a sulfonate group, a phosphate group, or a phosphonate group. Different types of groups may also be used in the polymer having a net negative charge. Preferably, the polymer having a net negative charge in solution comprises carboxylate groups. Most preferably, the polymer having a net negative charge is a polymer having one pendant carboxylate group per 2 to 10 carbon atoms of the backbone of the polymer.

[0036] Suitable polymers to be used in the process according to the invention are for example poly(styrene sulfonate) (PSS), poly(vinyl sulfonate) (PVS), dextran sulphate (DS), carboxymethyl dextran (CMD), carboxymethyl cellulose (CMC) and poly(acrylic acid) (PAA), or copolymers thereof. Preferably, the polymer having a net negative charge in solution is poly(acrylic acid) or a copolymer thereof.

[0037] The polymer having a net negative charge may comprise functional groups that can be used for reaction with a compound suitable for selectively binding an analyte of interest. Examples of such functional groups include carboxylic acid, amine, azide, alkene, alkyne, thiol, maleimide, or biotin.

[0038] The molecular weight of the polymer having a net negative charge in solution may vary between wide ranges, and is for example between 2 and 5000 kDa, preferably between 50 - 4000 kDa, and more preferably between 200-600 kDa. In this range, the thickness of the polymer coating on the SIN surface is higher. When using the object according to the invention in a biosensor, more negatively charged groups that can be used to attach receptors (directly or via a linking group).

[0039] The concentration of the polymer having a net negative charge in the polymer solution may also vary between wide ranges, and lies for example between 0.01 and 20 g / l, preferably between 1 g / l - 10 g / l. In this range, the thickness of the polymer coating on the SIN surface is higher. When using the object according to the invention in a biosensor, more negatively charged groups that can be used to attach receptors (directly or via a linking group).

[0040] The solvent present in the polymer solution comprising a polymer having a net negative charge may vary. For example, water or dimethylformamide may be used. Preferably, the polymer having a net negative charge is dissolved in an aqueous solution.

[0041] The pH of the polymer solution may affect the charge of the polymer and the charge of the SiN and / or SiO surfaces. Any pH whereby the SiN surface is selectively modified in the process according to the invention is a suitable pH. The pH of the polymer solution in the process according to the invention may be optimized for a given polymer.

[0042] For a solution comprising a polymer comprising carboxylate groups, the pH of the polymer solution preferably ranges between 1 and 6, preferably the pH of the polymer solution ranges between 2 and 4.

[0043] In the step of the process according to the invention wherein the etched object is treated with a polymer solution comprising a polymer having a net negative charge to obtain an object having a polymer coating on the at least one SiN surface, a salt may be present. The salt is preferably a salt that dissociates completely in water. Preferably the salt is an inorganic salt. More preferably, the salt is NaCI.

[0044] The concentration at which the salt may be present may vary between wide limits. The salt may for example be present in a concentration between 0.01 and 1 M, preferably between 0.1 and 0.4 M.

[0045] In the process according to the invention, the object having a polymer coating on the at least one SiN surface, is optionally irradiated to obtain an object having a crosslinked polymer coating on the at least one SiN surface. Crosslinking of polymers using irradiation is well known. It will depend on the polymer used in the coating what suitable irradiation conditions are. A person skilled in the art can easily identify suitable radiation dosages, and add e.g. initiators if needed. A suitable irradiation source is e.g. UV light.

[0046] In the process according to the invention, the object having a (crosslinked) polymer coating on the at least one SiN surface, is optionally treated with a silane to obtain an object with a silane coating on the at least one SiO surface. Modification of oxide surfaces with silanes is known.

[0047] Suitable silanes are for example hydrosilanes or hydrolysable silanes. Examples of hydrosilanes are RaSiH, RaSiHa, RSiHa, wherein R denotes a hydrocarbylgroup, optionally containing heteroatoms and wherein each R group may be the same or different. Hydrosilanes of the formula RaSiH are preferred.

[0048] In an embodiment, the hydrosilane may be represented by

[0049] Ra

[0050] Rb- Si - Rc

[0051] Rdwhere at least one of Ra, Rb, Rc, and Rd is H and at least one of Ra, Rb, Rc, and Rd is not H, where each of Ra, Rb, Rc, and Rd is, independently,

[0052] H, linear C1-30 alkyl, branched C1-30 alkyl, cyclic C3-30 alkyl, linear C2-30 alkenyl, branched C2-30 alkenyl, linear C2-30 alkynyl, branched C2-30 alkynyl, C6-20 aralkyl, C6-10 aryl, or a polymeric moiety having a molecular weight of about 1000 to about 100,000. The polymeric moiety is selected from the group consisting of hydrocarbon polymers, polyesters, polyamides, polyethers, polyacrylates, polyurethanes, epoxides, polymethacrylates and polysiloxanes (e.g. poly(methylhydrosiloxane)). Each of Ra, Rb, Rc, and Rd is optionally substituted with one or more substituents selected from the group consisting of — F, — Cl, — Br, — CN, — NO2, =0, — N=C=O, — N=C=S,

[0053] — N3, — NReRf, — SRg, — ORh, — CO2Ri, — PRjRkRI, — P(ORm) (ORn) (ORp), — P(=O) (ORq) (OR8), — P (=0)20Rt, — OP(=O)2ORu, — S(=O)2Rv, — S(=O)Rw, — S(=O)2ORx, — C(=O)NRyRz, and — OSiRaaRbbRcc. Each of Re, Rf, Rg, Rh, Ri, Rj, Rk, RI, Rm, Rn, Rp, Rq, Rs, Rt, Ru, Rv, Rw, Rx, Ry, and Rz, is, independently, H, linear C1-10 alkyl, branched C1 -1 Oalkyl, cyclic C3-8 alkyl, linear C2-10 alkenyl, branched C2-10 alkenyl, linear C2-10 alkynyl, branched C2-10 alkynyl, C6-12aralkyl, or C6-10 aryl, and is optionally substituted with one or more substituents selected from the group consisting of — F, — Cl, and — Br. Each of Raa, Rbb, and Rcc is, independently, linear C1-10 alkyl, branched C1-10 alkyl, cyclic C3-8 alkyl, linear C2-10 alkenyl, branched C2-10 alkenyl, linear C2-10, alkynyl, branched C2-10 alkynyl, C6-12 aralkyl, C6-10 aryl, — F, — Cl, — Br, or ORdd, where Rdd is linear C1-10 alkyl or branched C1-10 alkyl.

[0054] Hydrosilanes of the formula RaSiH are preferred. Preferred hydrolysable silanes are for example alkoxysilanes or chlorosilanes.

[0055] The object used in the process according to the invention is preferably a photonic chip, preferably a photonic sensor chip, wherein the SiN surface is part of an optical waveguide structure. Suitable photonic chips are e.g. chips that comprise a SiN planar optical waveguide embedded in a SiO cladding, which cladding has been partially removed, to allow modification of the SiN surface and optionally SiO surface.

[0056] Such photonic chips are known, and for example described in Besselink et al - biosensors 12 (2022) 553], hereby incorporated by reference.

[0057] When the object is a sensor chip, the (optionally crosslinked) polymer coating on the at least one SiN surface has been modified with a compound suitable for selectively binding an analyte of interest, which makes it possible to detect and / or quantify such analyte.

[0058] Thus, a sensor chip is defined herein as a chip on which at least one SiN surface has been modified with a compound suitable for selectively binding an analyte of interest, A photonic sensor chip is defined herein as a sensor chip that comprises a SiN planar optical waveguide embedded in a SiO cladding, which cladding has been partially removed, to allow modification of the SiN surface and optionally of the SiO surface, and wherein at least one SiN surface has been modified with a compound suitable for selectively binding an analyte of interest.

[0059] Thus, the invention also relates to sensor chips obtainable by the process according to the invention.

[0060] In the framework of this invention, the term “compound suitable for specifically binding an analyte of interest” is used interchangeably with the term “receptor”. Such a receptor may be for example be a peptide, a protein, an antibody, a nucleic acid (DNA or RNA), an aptamer, an enzyme, a DNAzyme, or a molecularly imprinted polymer (MIP). The receptor may be attached directly to the (optionally crosslinked) polymer coating, or via linking groups. For example, if the (optionally crosslinked) polymer comprises carboxylate groups and the receptor comprises amine groups, the carboxylate groups may first be activated by reaction with of N-ethyl-N'-3- (dimethylaminopropyl)carbodiimide (EDC) / N-Hydroxysuccinimide (NHS), followed by reaction with amine groups of the receptors.

[0061] Another example is to use the negatively charged polymer coating as a substrate for the adsorption of further polymer layers, e.g. a positively charged polymer such as poly-L-lysine- graft-poly(ethylene glycol), a copolymer commonly known as PLL-PEG. Receptors may be attached to the PEG chains either before or after adsorption of PLL- PEG to the polymer coating. Other positively charged polymers that may be adsorbed to the negatively charged polymer coating include e.g. poly-L-lysine (PLL), poly(ethyleneimine) (PEI) and other polymers comprising amine groups. Adsorption of a positively charged polymer may again be followed by adsorption of a negatively charged polymer, e.g. PAA or carboxymethyldextran (CMD). The process of alternating adsorption of positively and negatively charged species may be repeated several or many times. This iterative process is known as layer-by-layer assembly.

[0062] In a preferred embodiment, the silane of step d) comprises an anti-biofouling moiety, preferably a zwitterionic moiety or a non-ionic hydrophilic moiety. The presence of an ant-biofouling moiety reduces biofouling. In the framework of the invention, biofouling is defined as unwanted adsorption of molecules. The only desired adsorption is the binding of the receptors to the polymer coating on the SiN surface (which may be done directly or indirectly, e.g. using linker groups or intermediate layers as described above), binding of the analyte of interest to the receptors present on the polymer coating on the SiN surface, and optionally the binding of other reagents to the analyte of interest.

[0063] Examples of anti-biofouling moieties are zwitterionic moieties or non-ionic hydrophilic moieties. Examples of suitable zwitterionic moieties are polysulfobetaines, polycarboxybetaines, polyphosphobetaines, or polyampholites. Examples of suitable non-ionic hydrophilic moieties are ethylene glycol, propylene glycol, or glycerol, or oligomers of (2-hydroxyethyl)acrylate, (2-hydroxyethyl)methacrylate, (2- hydroxyethyl)acrylamide, (2- hydroxyethyl)methacrylamide. (2-hydroxypropyl)acrylate, (2-hydroxypropyl)methacrylate, (2-hydroxypropyl)acrylamide, (2-hydroxypropyl)methacrylamide, N- isopropylacrylamide, 2- methyl-2-oxazoline, 2-ethyl-2-oxazoline, N-vinylpyrrolidone, N-vinyl acetamide, vinyl alcohol, vinyl acetate, vinyl butyral, or amino acids.

[0064] In the framework of this invention, non-specific adsorption is defined as adsorption of all compounds other than the analyte of interest on receptors present on the polymer coating on the SiN surface. The receptors may be present either directly on the polymer coating or via linking groups or via intermediate layers. The chemistry to couple the desired receptors to the polymer coating on the SiN surface directly or indirectly, e.g. via linking groups or via intermediate layers is well known.

[0065] Analytes of interest may be but are not limited to peptides, proteins, nucleic acids (DNA, RNA), cells or cell fragments, extracellular vesicles, carbohydrates, fatty acids, hormones, or metabolites.

[0066] Sensor chips can be used in (bio)sensors. A (bio) sensor is defined herein as a device comprising at least one sensor chip, preferably a photonic sensor chip. When the analyte of interest is a biological analyte such as a biomolecule (e.g., a peptide, a protein, a nucleic acid, a carbohydrate, a fatty acid, a hormone, or a metabolite), a cell or a cell fragment or an extracellular vesicle, a virus, a bacterium, or the like, a sensor may be referred to as a biosensor. Thus, the invention also relates to (bio)sensors comprising a sensor chip obtainable by the process according to the invention. In a preferred embodiment, a sensor chip according to the invention is a sensor chip comprising at least one SiN and at least one SiO surface and comprising a crosslinked polymer coating on the SiN surface, preferably the crosslinked polymer coating is crosslinked poly(acrylic acid).

[0067] It is an advantage of process according to the invention that it allows the provision of objects comprising at least one SiN surface and at least one silicon oxide (SiO) surface with a (optionally crosslinked) polymer coating comprising a polymer with a net negative charge, which objects can be used in biosensors.

[0068] In particular objects according to the invention comprising a crosslinked polymer coating show a decreased non-specific binding and an improved specific binding, relative to objects coated with the same polymer which was not crosslinked.

[0069] Examples

[0070] The experiments described below illustrate various embodiments of the invention, without being limited thereto.

[0071] Materials and methods

[0072] Substrates

[0073] Patterned SisN^SiCh (SiN-SiO) substrates were obtained from LioniX International BV. Substrates are made from a 4 inch silicon wafers (single side polished) covered with an 8 micron thermal silicon oxide layer. On top of this a 100nm layer of SisN4 is deposited via Low Pressure Chemical Vapor Deposition (LPCVD). Patterning is done by dry etching of SisN4 with a mask to expose the underlying SiO2. Finally, the wafer was diced into 10x10 mm2substrates which consist of two large areas of 8x10 mm2(one area SiN, the other one SiO), and a smaller area of 2x10 mm2with alternating SiO and SiN lines with a width of 100 microns.

[0074] Chemicals

[0075] 4-Arm poly(ethylene glycol), hydroxy-terminated ([PEG-OH]4, Mw 20kD) was purchased from Creative PEGWorks. Poly(acrylic acid) (PAA, Mw 450kD), Poly(acrylamide-co-acrylic acid) partial sodium salt (PAA / PAAm = 9:1 Mw. 520kD), Poly(styrene sulfonate) (PSS, 200kD), Poly(vinyl sulfonate) (PVS, Mw 170kD), Poly(styrene sulfonate-co-maleic acid) (PSS-co-PMicA, 20kD), Dextran sulfate (DS, Mw 40kD), Carboxymethyl cellulose (CMC, Mw 250kD), poly(ethylene glycol), methoxy-terminated (PEG-OMe, Mw 100kD), dimethylformamide (DMF), sulfuric acid 95-97% (H2SO4), and sodium chloride (NaCI) were purchased from Sigma-Aldrich. Poly(acrylamide-co-acrylic acid) (PAA / PAAm = 9:1 , random, Mn 140.000), Poly(acrylamide-co-acrylic acid) (PAA / PAAm = 6:4, random Mn 160.000) were purchased from Polymer Source Inc. Poly-(L-lysine)-poly(ethylene glycol) (PLL-PEG (PLL150-g(30)-MPEG2K), Mw 108kD) was purchased from Nanosoft polymers.

[0076] Carboxymethyl dextran (CMD, Mw 500kD) was purchased from TdBLabs. Hydrofluoric acid (HF, 5% VLSI Selectipur) was purchased from VWR. Acetone (semiconductor grade) was purchased from Alfa Aesar. All chemicals were used as received.

[0077] Equipment

[0078] Ultrapure water (18.2 MO. cm at 25°C was prepared using the Puranity TU3 UV / LIF+ system (VWR International). The pH was measured with Mettler Toledo FiveEasy™ FE20. pH adjustments were done by the addition of either 1M HCI or 1M NaOH. A Diener-ATTO Plasma Cleaner was used for the activation step.

[0079] Activation and etching of the substrate

[0080] Substrates were rinsed with acetone and water, followed by different (combinations of) activation and etching processes.

[0081] Acetone rinsing: substrates were rinsed with acetone, sonicated in acetone (3 min, 80 Hz, 100% power), and blown dry with a stream of nitrogen gas. Substrates were then rinsed with water and blown dry with a stream of nitrogen gas.

[0082] Plasma cleaning: Substrates were treated in the plasma cleaner (0,3 mbar 02(g), 5 min).

[0083] Piranha cleaning: Piranha solution was prepared by adding a solution of 50wt% H2O2 to concentrated H2SO4 in a ratio of 1 :3. Substrates were submersed in a fresh piranha solution for 5 minutes, after which they were thoroughly rinsed with water, followed by blow drying with a stream of nitrogen gas.

[0084] Treatment with a hydrogen fluoride solution: A 1 wt% aqueous solution of HF was prepared by dilution from a 5% HF stock solution. Substrates were submerged for 90s, after which the etched substrates are thoroughly rinsed with water and blow dried with a stream of nitrogen gas.

[0085] Table 1: Comparison of HF etching times. It was found by XPS analysis that after etching for 60 s using 1 wt% HF, 2.5 atom% of carbon was still present on the SiN surface (see

[0086] Table 1). After etching for 90 s, no measurable amount of carbon was detected anymore. Therefore, it was decided to use a 1 wt% HF solution and a treatment time of 90 s for all subsequent experiments.

[0087] Polymer adsorption

[0088] Etched substrates were directly submerged in a solution of the polymer. After polymer adsorption, the substrates having a polymer coating on the SiN surface were rinsed with water and sonicated subsequently in methanol and in water (10min, 80Hz, 100% power). The resulting substrates were dried with a stream of nitrogen.

[0089] UV irradiation

[0090] Substrates having a polymer coating on the SiN surface were placed in a quartz UV reactor. Air was removed by performing 3 cycles of evacuation, followed by filling of the reactor with argon. The reactor was closed under argon atmosphere, placed under the UV lamp (a Hg / Xe lamp equipped with a 260 nm mirror set, resulting in an collimated irradiation beam with an intensity of 10-15 mW / cm2 at 260 nm) and irradiated for 10 minutes.

[0091] Characterization by X-ray Photoelectron Spectroscopy (XPS)

[0092] XPS measurements were performed using a JPS-9200 photoelectron spectrometer (JEOL Ltd., Japan). All the samples were prepared and stored under ambient conditions prior to analysis using a focused monochromatic Al Ka X-ray source (spot size of 300 pm) radiation at 12 kV and 20 mA with an analyzer energy pass of 10 eV at a takeoff angle <|) (angle between sample and detector) of 80°. XPS wide-scan and narrow-scan spectra were obtained under UHV conditions (base pressure 3 x 10"7Torr). All narrow-range spectra were corrected with a linear background before fitting. The spectra were fitted with symmetrical Gaussian / Lorentzian [GL(30)] line shapes using CasaXPS. All spectra were referenced to the C 1s peak attributed to C-C and C-H atoms at 285.0 eV.

[0093] The atomic C / Si ratios were converted into monolayer thicknesses (dMi.) using the following relationship [X. Wallart, C. H. de Villeneuve, P. Allongue, J. Am. Chem. Soc., 2005, 127, 7871-7878]: CIML( ) = AS'ML X sin(<t>) x Ln(1 + C / Si) were AS'ML is the attenuation length of Si2Pphotoelectrons in the organic monolayer (ASiML= 39,5 A).

[0094] Examples of processes which do result in a polymer layer with a thickness on SiN greater or equal than 0.5 nm and a polymer layer thickness on SiO equal to or less than 0.1 nm are embodiments of the invention and are labelled as Examples (Ex.). Experiments that do not meet these criteria are labelled as Comparative Experiments.

[0095] Standard Protocol

[0096] Where a protocol is denoted as “standard protocol”, the following combination of process parameters was used: oxygen plasma activation, etching with a 1 wt% HF solution for 90 seconds, treatment with a 5 g / l poly(acrylic acid) solution in water, at pH 3, for 15 minutes, using a poly(acrylic acid) with a molecular weight of 450 kDa, the solution having a 0.3 M NaCI concentration.

[0097] Examples (Ex.) and Comparative Experiments (Cex)

[0098] Comparison of different activation and etching processes

[0099] Samples were activated and / or etched (see general procedures for details) and subsequently submerged in a 5 g / l solution of PAA (450 kDa) in 0.3 M NaCI, pH 3 for 15 minutes. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in table 2.

[0100] Table 2: Comparison of different activation and etching processes.

[0101] The results in Table 2 show that the process according to the invention may comprise different (combinations of) activation and etching treatments. The activation and / or etching process removes organic contaminations and provides a clean, hydrophilic surface. This treatment results in reproducible surface properties for subsequent surface modification by PAA, and an increased thickness of the PAA layer on SiN. Molecular weight of PAA

[0102] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, samples were immersed in a 5 g / l solution of PAA in 0.3 M NaCI, pH 3. The molecular weight of the PAA was varied. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in table 3. Table 3: Comparison of different PAA molecular weights.

[0103] Examples 7-10 show that selective surface modification of SiN occurs using a wide range of PAA molecular weights. Therefore, the invention is not particularly limited by the molecular weight of the polymer.

[0104] Concentration of PAA

[0105] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, etched substrates were immersed in a solution of PAA (450 kDa) in 0.3 M NaCI, pH 3. The concentration of the PAA solution was varied. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in Table 4. Table 4: Comparison of different PAA concentrations. Examples 6 and 12-16 show that selective surface modification of SiN occurs using a wide range of PAA concentrations. At the lowest used concentration, selective surface modification did not occur within 15 minutes (experiment 11). Extending the adsorption time to 22 hours (experiment 12) results in increased absorption on the SiN surface, but also increases the adsorption on SiO. Thus, a wide range of polymer concentrations higher than 0.001 g / l may be used in step b) of the process according to the invention. pH

[0106] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, etched substrates were immersed in a 5 g / l solution of PAA (450 kDa) in 0.3 M NaCI. The pH of the solution was varied. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in Table 5.

[0107] Table 5: Comparison of different pH values.

[0108] Experiments 6 and 17-20 show that selective surface modification of SiN occurs over a wide range of pH values, with pH 3 giving the highest layer thickness on SiN. Lower and higher pH values also give good results, but at pH 8.5 the thickness of the PAA layer on SiN becomes too small. This is attributed to the fact that at such high pH values the SiN surface becomes negatively charged, thus resulting in reduced adsorption of negatively charged PAA. .

[0109] NaCI concentration

[0110] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, etched substrates were immersed in a 5 g / l solution of PAA (450 kDa), pH 3. The NaCI concentration of the solution was varied. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in Table 6.

[0111] Table 6: Comparison of different NaCI concentrations.

[0112] Experiments 6 and 21-24 show that selective surface modification of SiN occurs over a wide range of NaCI concentrations.

[0113] Copolymers of PAA with Poly(acrylamide) (PAAm)

[0114] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, etched substrates were immersed in a 1 g / l solution of a random PAA- PAAm copolymer in 0.3 M NaCI, pH 3.5. The ratio between PAA and PAAm monomers in the copolymer was varied. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in Table 7.

[0115] Table 7 Comparison of PAA-PAAm copolymers.

[0116] Experiments 16 and 25-27 show that the invention is not limited to homopolymers. PAAm is a charge neutral monomer, so these experiments also show that not all monomers in the polymer have to be negatively charged or chargeable.

[0117] Non-aqueous solvents

[0118] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, etched substrates were immersed in a 5 g / l solution of PAA in different solvents. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in Table 8.

[0119] Table 8: Comparison of different solvents.

[0120] Experiments 29 and 30 show selective surface modification of SiN can also be achieved when non-aqueous PAA solutions are used. The invention is therefore not limited to aqueous polymer solutions. Polymers other than PAA

[0121] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, etched substrates were immersed in a 5 g / l polymer solution in 0.3 M NaCI, pH 3.5. The type of polymer was varied, see Table 9. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. The thickness of the surface modification layer on SiN and SiO was measured by XPS as described above. The results are shown in table 10. Table 9: Polymers used for experiments 30-38. Table 10: Comparison of different polymers.

[0122] Experiments 30-35 show that selective surface modification of SiN can be achieved using different polymers with negatively charged or chargeable groups.

[0123] Experiments 36-38 show that if the polymer does not comprise any negatively charged or chargeable groups, no selective surface modification of SiN is observed. In particular, experiment 37 shows that if the positively charged polymer PLL-PEG is used, the opposite selectivity is achieved, i.e., the thickness of the surface modification layer on SiN is significantly smaller than on SiO. These experiments therefore show that it is an essential feature of the invention that the polymer in solution is a polymer having a net negative charge. The polymer as such, i.e not in solution, does not need to have negatively charged groups, but in solution negatively charged groups should be present.

[0124] UV crosslinking of PAA and surface modification of SiO

[0125] For these experiments, a photonic sensor chip was used as the substrate. The chip comprises a SiN planar optical waveguide embedded in a SiO cladding. The cladding has been partly removed to expose the surface of the SiN waveguide to the sample solution. The chip therefore comprises a SiN and a SiO surface. For biosensing applications, the SiN surface needs to be modified to immobilize compounds which can recognize and bind the analyte of interest (also called bioreceptors). On the other hand, adsorption of biomolecules on the SiO surface (biofouling) has to be prevented as much as possible.

[0126] Substrates were rinsed with acetone and water, followed by plasma activation and HF etching. Next, etched substrates were immersed in a 5 g / l solution of PAA (450 kDa) in 0.3 M NaCI, pH 3. After 15 minutes, substrates were removed from the solution, rinsed with water and dried with a stream of nitrogen. Subsequently, the PAA coating on SiN was crosslinked by irradiation with UV light (conditions??). Finally, the SiO surface was modified by treatment with a PEG-silane.

[0127] Details about the chip, the measurement set-up and the measurements themselves have been described in Besselink et al, Biosensors 12 (2022) 553.. Briefly, waveguides on the chip are configured in such a way that they constitute an asymmetric Mach- Zehnder interferometer. The presence of biomolecules on the exposed part of the SiN waveguide results in a phase shift of the interference pattern at the output of the sensor. This phase shift is the measured sensor signal, which is expressed in picometers (pm), and which is proportional to the amount of biomolecules present on the waveguide.

[0128] Non-specific adsorption of BSA (bovine serum albumin) was measured for a UV irradiated and a non-UV-irradiated sensor chip. The result of this experiment is shown in

[0129] Table 11. For a non-UV-irradiated PAA layer (experiment 39), a signal of 52096 pm was measured. For a UV-irradiated PAA-layer (experiment 40), a signal of only 2757 pm was measured. Thus, UV irradiation greatly decreases the non-specific adsorption of BSA, in this case by approximately 95%.

[0130] Table 11: Effect of UV crosslinking of PAA on non-specific adsorption.

[0131] Because there is less non-specific binding of proteins to the waveguide if the PAA layer is crosslinked, the number of available binding sites for analytes is increased.

[0132] Moreover, the crosslinked PAA layer does not swell and is therefore thinner. This means that analytes will bind closer to the waveguide, where the optical sensitivity is the highest. Therefore, UV crosslinking of the PAA layer is expected to result in an increased specific sensor signal.

[0133] To demonstrate this effect, the Spike Protein (SP) of the Sars-CoV-2 virus was immobilized on the SiN waveguide by EDC / NHS coupling to the carboxylic acid groups of the PAA layer as described in [besselink - biosensors 12 (2022) 553], Then, the sensor was exposed to 4% serum (serum was 25x diluted with buffer) without Sars- CoV-2 antibodies (negative control, sensor signal 1 in Table 12), followed by exposure to 4% serum containing 4 pg / ml of a polyclonal rabbit anti-SP antibody (primary binding step, sensor signal 2 in Table 12). Finally, the sensor was exposed to 10 pg / ml of a fluorescently labelled goat-anti-rabbit antibody to amplify the signal (secondary binding step, sensor signal 3 in Table 12).

[0134] Table 12: Effect of UV crosslinking of PAA and PEG modification of SiO on analyte detection.

[0135] The results of a sensor with (experiment 41) and without (experiment 42) UV crosslinking of the PAA layer are compared in Table 12. The negative control (sensor signal 1) measures non-specific adsorption of serum proteins on the waveguide. The result shows that there is less non-specific adsorption the crosslinked PAA layer, thus confirming the results from experiments 39 and 40. On the other hand, the sensor with the crosslinked PAA layer shows much higher specific binding signals in the primary and secondary binding steps (sensor signals 2 and 3), thus showing that UV crosslinking has a positive effect on the performance of the sensor.

Claims

CLAIMS1. A process for selective surface modification of an object comprising at least one SiN surface and at least one silicon oxide (SiO) surface, the process comprising the steps of a. Treating the object with hydrogen fluoride (HF) solution to obtain an etched object; b. Treating the etched object with a polymer solution comprising a polymer having a net negative charge to obtain an object having a polymer coating on the at least one SiN surface; c. Optionally irradiating the object having a polymer coating on the at least one SiN surface with ultraviolet light to obtain an object having a crosslinked polymer coating on the at least one SiN surface; d. Optionally treating the object having a (optionally crosslinked) polymer coating on the at least one SiN surface with a silane to obtain an object with a silane coating on the at least one SiO surface.

2. The process according to claim 1, wherein prior to step a) the object has undergone an activation treatment.

3. The process according to claim 2, wherein the activation treatment comprises an oxidation treatment, preferably exposure to air plasma or oxygen plasma, piranha solution, or UV / ozone, more preferably exposure to air plasma or oxygen plasma.

4. The process according to any of the preceding claims, wherein the treatment with a HF solution comprises exposing the object to an aqueous HF solution with a concentration between 0.5 and 3 wt% HF, relative to the weight of the HF solution.

5. The process according to any of the preceding claims, wherein the polymer having a net negative charge comprises negatively charged groups selected from a carboxylate group, a sulfonate group, a phosphate group, or a phosphonate group, most preferably a carboxylate group.

6. The process according to claim 5, wherein the negatively charged groups are part of a pendant group.

7. The process according to any of the preceding claims, wherein in step b) a salt is present, preferably a salt that dissociates completely, more preferably an inorganic salt , preferably NaCI.

8. The process according to claim 7, wherein the salt is present in a concentration between 0.01 and 1 M, preferably between 0.1 and 0.4 M.

9. The process according to any of the preceding claims, wherein the polymer having a net negative charge is selected from poly(acrylic acid) (PAA), poly(methacrylic acid), carboxymethyl dextran, carboxymethyl cellulose, hyaluronic acid, poly(sodium-4-styrene sulfonate), poly(vinyl sulfonate), poly(vinyl phosphoric acid), or cellulose phosphate, or copolymers thereof.

10. The process according to any one of the preceding claims, wherein the polymer having a net negative charge is poly(acrylic acid).11 . The process according to any one of the preceding claims, wherein the polymer having a net negative charge has a molecular weight between 2 and 5000 kDa, preferably 50 - 4000 kDa, more preferably 200-600 kDa.

12. The process according to any one of the preceding claims, wherein the polymer solution comprising the polymer having a net negative charge has a polymer concentration between 0.01 and 20 g / l, preferably 1 g / l and 10 g / l.

13. The process according to any one of the preceding claims, wherein the pH in step b) is or is adjusted to a value between 1 and 6, preferably between 2 and 4.

14. The process according to any one of the preceding claims, wherein polymer having a net negative charge is dissolved in an aqueous solution .

15. The process according to any one of the preceding claims, wherein the polymer comprising the net negative charge is poly(acrylic acid), and wherein the poly(acrylic acid) is subsequently crosslinked by using UV irradiation.

16. The process according to any one of the preceding claims, comprising step d) and wherein the silane of step d) is a hydrosilane or a hydrolysable silane, and wherein hydrolysable silanes are preferably selected from alkoxysilanes or chlorosilanes.

17. The process according to claim 16, wherein the silane of step d) comprises an antibiofouling moiety, preferably a zwitterionic moiety or a non-ionic hydrophilic moiety.

18. The process according to any one the preceding claims, wherein the process comprises the step of functionalizing the polymer coating with a compound suitable for specifically binding an analyte of interest.

19. The process according to any one of the preceding claims, wherein the object is a sensor chip, preferably a photonic sensor chip, wherein the SiN surface is part of an optical waveguide structure.

20. A sensor chip obtainable by the process according to any one of the preceding claims.21 . A sensor chip comprising at least one SiN and at least one SiO surface and comprising a crosslinked polymer coating on the SiN surface.

22. A sensor chip according to claim 21 wherein the crosslinked polymer coating is crosslinked poly(acrylic acid).

23. A biosensor comprising an object obtainable by the process according to any one of claims 1-18, wherein the object is the sensor chip .

24. A biosensor comprising a sensor chip according to any one of claim 20-23.

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