Chemistry

Easy Floor Modification of Poly(dimethylsiloxane) through Floor Segregating Good Polymers for Biomicrofluidics

Floor modification of PDMS with PDMS-PEG BCP components

We chosen a PDMS-PEG BCP because the sensible copolymer additive for hydrophilizing the PDMS floor. This copolymer, a commercially out there surfactant (Gelest, product code DBE-712), features a hydrophobic PDMS section appropriate with the bottom elastomer (e.g. PDMS) and a hydrophilic, fouling resistant PEG block. Its molar mass is 600, and accommodates 60–70% PEG. The PDMS section solubilizes the additive inside the elastomer matrix throughout preparation after which anchors the additive within the cured PDMS. It will also be linked with the bottom PDMS through the plasma remedy used for bonding the system collectively, bettering the longevity of the floor modification. The brief chain size and BCP structure of the additive results in its segregation to the pattern floor18,27. When the pattern floor is uncovered to water (e.g. when the microfluidic channel is full of aqueous media), the copolymer self-organizes on the PDMS/water interface to show the PEG segments to the aqueous resolution and create a steady hydrophilic floor that forestalls the adsorption of proteins and different bio-macromolecules (Fig. 1) with out utilizing any further steps or altering the manufacturing course of.

Determine 1Figure 1

Schematic diagram of the PDMS floor modification technique. PDMS and the PDMS-PEG BCP components are blended, and the system is fabricated following common processes (no added steps). The copolymers segregate to the PDMS floor in air. When in touch with water, floor rearrangement creates a floor lined with PEG teams that forestall non-specific adsorption of proteins and permits the circulate of polar liquids.

So far, most approaches, to cut back hydrophobicity of PDMS, have been developed utilizing post-treatment methods8,23,28,29,30,31 that add a number of new, cumbersome steps to the micro-device manufacturing course of, usually requiring particular tools and/or hazardous routes9. This renders them unfavorable for large-scale fabrication and for adoption by a large consumer base. Additional, they can’t be tailored to the manufacture of different silicone-based elastomers (e.g. tubing, seals). This limits their impression. The method we current right here is differentiated by its simplicity throughout use, in contrast with different approaches that depend on coatings or post-processing (Desk 1).

Whereas a handful of research have utilized surface-segregating amphiphilic copolymers to enhance the floor hydrophilicity of PDMS, none have demonstrated excessive levels of hydrophilicity with out loss in mechanical properties and/or optical readability (Desk 1). Moreover, these research have nearly completely targeted on characterizing surfaces that haven’t been subjected to the total slew of processes concerned in microfluidic system manufacture, together with an alcohol soak for disinfection and plasma remedy for bonding of the system. These processes can leach these components and/or considerably alter floor chemistry. As well as, not one of the previous research characterize the viability of cells upon publicity to those PDMS blends. These amphiphilic components might leach into the feed going by means of a biomicrofluidic system, killing cells and thus rendering these approaches moot in sensible settings. Thus, there’s a important data hole in not solely creating novel components for PDMS for floor modification but additionally in higher understanding their habits all through the life cycle of a biomicrofluidic system.

Hydrophilicity and wettability of PDMS with PDMS-PEG BCP components

To check our speculation that the PDMS-PEG BCP additive would result in elevated hydrophilicity that is still steady over lengthy timescales, we measured sessile drop water contact angles (WCA) on PDMS-PEG BCP modified PDMS surfaces and in contrast them to the unmodified PDMS over a 20-month period. We used dynamic contact angle measurements, that are helpful for evaluating the wettability and hydrophilicity of modified PDMS surfaces32,33. Determine 2a reveals the variation of the WCA of PDMS samples ready with various quantities of PDMS-PEG BCP additive in time. The preliminary contact angles of all samples (besides the one containing 2% PDMS-PEG BCP additive) have been fairly excessive, between 94–106°. This means that in air, the pattern floor is generally lined with hydrophobic PDMS segments. Nevertheless, whereas the WCA of PDMS with no PDMS-PEG BCP remained regular above 101° through the 45-minute experiments, the WCA of all PDMS with PDMS-PEG BCP components decreased in time. Moreover, this lower was usually proportional to the focus of PDMS-PEG BCP components. After 45 minutes of publicity to water, the PDMS-PEG BCP additive containing pattern surfaces grew to become considerably extra hydrophilic than additive-free PDMS. As little as zero.125% PDMS-PEG BCP additive led to a last contact angle of 69.6° (Supporting Data Fig. S1), comparable with the bottom contact angles reported for different additive-modified PDMS techniques20,21,22. The very best BCP containing samples (1.5% and a pair of% PDMS-PEG BCP) have been absolutely wetted (WCA ≈ zero°) in our dynamic measurements. Nonetheless, it is very important be aware that rising BCP focus for lowering hydrophobicity isn’t the one requirement for profitable and steady floor modification. We encountered bonding issues on glass slides throughout oxygen plasma remedy at increased copolymer concentrations (1.5 and a pair of (w/w %)), so we eradicated these concentrations for additional experiments.

Determine 2Figure 2

PDMS with PDMS-PEG BCP components dramatically reduces hydrophobicity. (a) The change in WCA with time for varied additive ratios (zero.125–2%), exhibiting polymer reorganization. (b) WCA of PDMS with/with out PDMS-PEG BCP components after soaking them IPA for 24 hours (AS) and treating them with O2 plasma (AS + PT) at totally different time intervals. (c) Remaining (t = 45 min) WCAs for various PDMS-PEG BCP additive ratios after 20 months of storage (with/with out plasma remedy) present the steadiness of the modified supplies, indicating that the samples ready with PDMS-PEG BCP components don’t lose their hydrophilic traits for a protracted interval. The info are proven because the imply ± SD (n = three).

These outcomes affirm that upon publicity to water, the PDMS-PEG BCP additive self-assembles on the interface to create a hydrophilic PEG layer, and point out that this rearrangement happens quicker and extra successfully with rising PDMS-PEG BCP content material. Moreover, this method can result in last WCA values a lot under earlier reviews for additive-modified PDMS supplies, which vary between 84° and 63°20,21.

In sensible functions, PDMS isn’t used instantly after molding. PDMS units are usually sterilized by immersion into an alcohol corresponding to IPA, which can leach out components. Then, they’re handled with O2 plasma and bonded to glass, a silicon wafer, or one other piece of PDMS. It’s essential for the improved floor hydrophilicity to be steady throughout these processing steps. Moreover, microfluidic units will not be essentially used instantly after manufacture. Subsequently, the modified floor must be steady over very long time durations.

To check these parameters, we first established the soaking time of PDMS with and with out PDMS-PEG BCP components in IPA. We measured the WCA of PDMS with out PDMS-PEG and of PDMS with zero.5% PDMS-PEG BCP additive after soaking in IPA for six, 12 and 24 hours (Supporting Data Fig. S2). The hydrophilicity of samples after 6 hours of IPA soaking was increased in contrast with that of samples with 12 hours and 24 hours of soaking. We imagine that in soaking in IPA, the decrease molar mass fractions of the PDMS-PEG BCP subtle out21. This resulted in a lower in copolymer focus within the PDMS and a major improve within the contact angle. We noticed that 12 hours of IPA soaking was enough to take away all of the decrease mass fractions of the BCP as a result of no important change in hydrophilicity was noticed between 12 hours and 24 hours IPA soaking. Nonetheless, some PDMS-PEG BCP remained within the PDMS as the ultimate contact angles have been nonetheless a lot decrease than that of PDMS with no PDMS-PEG BCP. The remaining PDMS-PEG BCP molecules have been possible of upper molar mass, which improved the long-term stability of the layer. Moreover, this decreased the danger of the additive, significantly low molar mass fractions prone to act as cytotoxic surfactants, leaching out of the PDMS throughout operation, which might negatively impression cell viability. Nonetheless, we chosen 24 h IPA soaking for experimental practicality and to make sure constant leads to additional experiments (samples labeled AS).

The improved floor hydrophilicity of IPA-soaked samples was steady for no less than 20 months (Fig. S3). A number of the samples have been then handled with O2 plasma (samples labeled AS + PT) (Fig. 2b). A day after O2 plasma remedy (AS + PT 1 d), the hydrophobicities of PDMS samples each with and with out PDMS-PEG BCP components have been considerably lowered (Fig. 2b). The WCA of PDMS with no PDMS-PEG BCP grew to become 63.three°.

It has beforehand been reported that PDMS surfaces uncovered to plasma exhibit elevated oxygen content material and probably silicon (Si) atoms are bonded to a few or 4 oxygen atoms and that this reduces hyrophobicity34. The principle problem posed by the plasma oxidation is the eventual hydrophobic restoration. This can be a results of the reorientation of pre-existing oligomers from the majority to the floor35. Certainly, three days after plasma remedy (AS + PT three d), the WCA of the PDMS with no PDMS-PEG BCP additive returned to its preliminary worth of 102°. PDMS with PDMS-PEG BCP components additionally exhibited a rise in hydrophilicity upon O2 plasma remedy. The surfaces grew to become absolutely wettable a day after plasma remedy with WCA values round zero°. As seen in Fig. 2b, though a minor improve in wettability was noticed, all PDMS with PDMS-PEG BCP components maintained their hydrophilicity, with WCA values between 54.2° ± 2.7° and 25.7° ± 2°. These values are considerably decrease than earlier reviews20,21,22. We imagine that the existence of PEG on the modified PDMS floor enhances Si-O bonding and consequently, extra SiOx-rich layer and extra hydrophilic surfaces could be obtained as in comparison with PDMS with no PDMS-PEG BCP. Importantly, this enhanced floor hydrophilicity was steady for no less than twenty months. The elevated floor hydrophilicity might have enhanced the floor segregation of the PDMS-PEG BCP by creating an area gradient, drawing the copolymer to the floor even earlier than publicity to water. The elevated diploma and stability of floor hydrophilicity can also be linked with the advanced and competing etching, deposition and response processes that happen throughout plasma remedy. Throughout the O2 plasma remedy, PDMS repeat items are partially etched on the floor, shedding their methyl teams and forming silica. The plasma remedy can also trigger cross-linking, however this impact is comparatively restricted in PDMS36. In distinction, oxygen-containing polymers corresponding to PEO are inclined to endure atomic re-arrangement reactions corresponding to cross-linking versus etching37. This suggests that the plasma remedy might preferentially etch the hydrophobic methyl teams from PDMS chains on the floor, exposing PEG segments that have been proper under. The plasma remedy can even chemically cross-link the PDMS-PEG BCP additive to the PDMS community. Moreover, it might result in cross-linking between PEO chains on the floor. This will anchor the PDMS-PEG BCP particularly on the highest floor of the pattern, bettering the longevity of floor modification. Moreover, PDMS samples with totally different PDMS-PEG BCP preserved their hydrophilic traits even after 20 months of storage (with/with out plasma remedy) indicating that samples ready with PDMS-PEG BCP components are steady for a protracted interval (Fig. 2c).

Characterization of the bodily properties of PDMS with PDMS-PEG BCP components

Transparency

Microfluidic units are generally used along with vibrant discipline and fluorescence microscopy for imaging cells38 to observe their well being and motility. Subsequently, supplies used for manufacturing such units have to be clear. Blue gentle [460–500 nm] excitation is often used to picture inexperienced fluorescent protein (GFP) and Calcein AM, and inexperienced gentle [528–553 nm] excitation is helpful for imaging crimson fluorophores38.

Accordingly, we assessed the optical readability of PDMS with and with out PDMS-PEG BCP components by measuring gentle transmittance by means of eight mm thick slabs between 400–600 nm wavelengths within the UV-visible vary earlier than and after an IPA soak (Supporting Data Fig. S4) after the fabrication. Transmittance values for the middle wavelengths of blue gentle (480 nm) and inexperienced gentle (540 nm) are given in Desk 2. Earlier than soaking in IPA (Fig. S4(a)), transparency values for PDMS with as much as zero.5% PDMS-PEG BCP have been akin to additive free PDMS, with all transmittance values above 96%. The transparency of the PDMS pattern with 1% PDMS-PEG was barely decrease, with transmittance values within the 80–88% vary. This will come up from the formation of micelles or comparable aggregates of the PDMS-PEG BCP surfactant inside the bulk PDMS at these increased concentrations, as noticed in different research20,21. After soaking in IPA (Fig. S4(b)), samples modified with zero.125% and zero.25% PDMS-PEG BCP components exhibited roughly the identical optical readability as unmodified PDMS. Nevertheless, the optical readability of modified PDMS with zero.5% and 1% PDMS-PEG BCP decreased, with transmittance values round 75% and 50%, respectively. PDMS samples containing zero.25% PDMS-PEG BCP efficiently mixed excessive optical readability with a hydrophilic floor.

Desk 2 Optical and mechanical properties of PDMS with PDMS-PEG BCP components.

Floor Characterization

We used X-ray photoelectron spectroscopy (XPS) to achieve a greater understanding of the adjustments in floor chemistry through the manufacture of biomicrofluidic units from PDMS with and with out the PDMS-PEG BCP additive. On this research, we targeted on PDMS with zero.25% PDMS-PEG BCP, chosen based on the standards described above, and PDMS with no PDMS-PEG BCP. We analyzed their floor chemistry at every stage of the microfluidic system manufacture course of. The fundamental floor compositions of each the PDMS with no PDMS-PEG and zero.25% PDMS-PEG, decided by the survey scan, remained primarily unchanged after soaking in IPA (Supporting Data Fig. S5). After plasma remedy, survey scans of PDMS with and with out PDMS-PEG BCP components each point out a rise in carbon and oxygen content material and a corresponding lower in silicon content material (Supporting Data Fig. S6). Excessive-resolution scans of C1s spectra have been used to achieve deeper perception into the chemical adjustments that occurred throughout these processes (Fig. three(a,b)).

Determine threeFigure 3

Excessive-resolution scans of C1s of each (a) PDMS with no PDMS-PEG BCP additive and (b) PDMS with zero.25% PDMS-PEG BCP additive earlier than IPA soaking (BS), after IPA soaking (AS), after IPA soaking and 1 day after O2 plasma remedy (AS + PT-1 d) and after IPA soaking and 1 week after O2 plasma remedy (AS + PT-1 wk) have been analyzed. Unmodified PDMS acquires hydrophilic teams upon plasma remedy, however these useful teams disappear from the floor inside per week. XPS information confirms that the PDMS-PEG BCP additive segregates to the floor, and creates hydrophilic teams on the floor upon plasma remedy.

PDMS with no PDMS-PEG spectra featured just one peak, at 284.2 eV, equivalent to C-Si bonds. This was unchanged upon soaking in IPA. Upon plasma remedy, peaks appeared at 286.three eV and 289.1 eV, assigned to C-O and C=O bonds, respectively39. These peaks, nonetheless, fully disappeared after per week. That is because of the restoration of hydrophobicity after oxidation by reorientation of the floor silanol teams into the majority polymer, which offers for the motion of free PDMS chains from the majority section to the floor and condensation of silanol teams on the surface8. In distinction, PDMS with zero.25% PDMS-PEG BCP confirmed each a robust peak close to 284.6 eV arising from C-Si bonds and a shoulder close to 286.three eV, equivalent to C-O bonds, indicating the presence of PEG segments from the PDMS-PEG BCP additive close to the floor. Upon plasma remedy, the depth of the C-O peak elevated, and a brand new peak equivalent to C=O appeared. These teams might come up each from reactions of PDMS and from reactions and cross-linking of PEG. Not like pure PDMS, the depth of the C-O and C=O peaks within the PDMS with zero.25% PDMS-PEG remained unchanged per week after plasma remedy. This phenomenon confirms the existence of PEG molecules on the modified floor for long-term stability after plasma remedy, which is in good settlement with the hydrophilicity information (Fig. 2a,b).

Mechanical properties

PDMS is an effective candidate to be used in microfluidic units resulting from its excessive compliance and adaptability. Its Younger’s modulus is determined by the precise formulation, and is round 1.32–2.12 MPa for the generally used pre-polymer to curing agent ratio of 10:140,41,42. Ideally, floor modification by any method mustn’t compromise these mechanical properties. To verify the mechanical properties of PDMS-PEG-modified PDMS samples, tensile energy and compressive modulus have been evaluated by dynamic mechanical evaluation (DMA) instantly after fabrication and likewise 20 months after fabrication. Younger’s modulus and compressive modulus of the modified samples have been calculated for the linear elastic area (<40% pressure). No important change was noticed with the mechanical properties of the PDMS-PEG BCP modified PDMS compared with literature research even after 20 months of storage.

Biocompatibility

Many microfluidic functions that make the most of PDMS and its alternate options contain tradition or circulation of cells from totally different tissues. Subsequently, when designing a brand new materials for biomicrofluidics, it’s essential to take its biocompatibility under consideration. As an example, the floor modifying components might leach from the system into the microfluidic channel and have an effect on cell viability and/or operate. This will result in poor system efficiency even when floor hydrophilicity is enhanced. So far, there are some research that evaluated the biocompatibility or cell adhesion of modified PDMS microfluidic units or slabs utilizing mammalian A549 cells43, L929 mouse fibroblasts44, tendon stem cells45, mesenchymal stem cells (MSCs)46, mind cerebral cortex cells47, HeLa cells48, and stroma cells49. To our data, not one of the earlier PDMS modification methods have been evaluated for compatibility with hepatocytes, the parenchymal cells of the liver, that are extremely prone to hostile reactions. The liver performs a central function in drug metabolism and detoxing so the event of liver-on-a-chip fashions for profitable prediction of poisonous response is on the heart of the current initiatives in the direction of in vitro human scientific trial approaches50,51. Right here, we used rat main hepatocytes to check the biocompatibility of our modified PDMS substrate in a easy microfluidic liver-on-a-chip mannequin.

To make sure that using the PDMS-PEG BCP in microfluidic system manufacture doesn’t adversely impression cell operate, we manufactured microfluidic units utilizing a glass backside and PDMS prime with or with out PDMS-PEG BCP components, and cultured main rat hepatocytes in these units. With a purpose to quantitatively consider cell viability, the cells have been stained with a dwell (inexperienced)/useless (crimson) stain three days after the tradition (Fig. four). Cells had excessive viability (>99.zero%) all through the three day tradition interval following the preliminary cell seeding into the microdevice. The usage of the PDMS-PEG BCP additive led to no seen or important variations in cell viability or morphology. PDMS-PEG modified microfluidic units carried out simply in addition to PDMS with no PDMS-PEG components and offered no hostile results. Since in vitro techniques are sometimes most popular as fashions to foretell drug toxicity and pharmacokinetics for scientific circumstances, this design could be simply scaled to create an array of in vitro research for speedy drug improvement or learning the toxicity of medication because of the simplicity of the system.

Determine fourFigure 4

Biocompatibility of PDMS with PDMS-PEG BCP components. Rat hepatocytes have been cultured in glass-(modified) PDMS units. No hostile results have been noticed (three days) with (a) PDMS with no PDMS-PEG and PDMS with (b) zero.125%, (c) zero.25%, (d) zero.5% and (e) 1% (w/w) PDMS-PEG BCP. Picture scale bar: 400 μm. Every experiment was carried out in triplicates from no less than three totally different rat isolations.

Protein adsorption on PDMS with PDMS-PEG BCP components

The principle purpose of creating this PDMS floor modification method was to create a fouling resistant floor and stop the non-specific adsorption of proteins onto the microfluidic system. That is motivated by two phenomena. First, a lot of the undesired bioreactions and bio-responses in synthetic supplies are promoted resulting from adsorbed proteins52,53. Second, many functions of biomicrofluidics contain controlling the publicity of cells to a recognized focus of a particular, desired protein corresponding to a biologic drug. Non-specific adsorption results in the lack of this drug by means of adsorption, exposing the cells to a decrease focus than presumed. This could result in a extreme underestimation of the toxicity and exercise of such medication. Whereas hydrophilicity is broadly correlated with decreased protein adsorption, the connection isn’t essentially simple54,55. Subsequently, we quantitatively measured the adsorption of two fluorescently-labeled proteins, albumin and lysozyme, on PDMS slabs with and with out PDMS-PEG BCP components (Fig. 5(a,b)), each instantly upon manufacture (Fig. 5a) and following processes that simulate biomicrofluidic system manufacture (Fig. 5b, IPA soak and 1 week after O2 plasma remedy).

Determine 5Figure 5

PDMS with PDMS-PEG BCP components exhibit decrease protein adsorption. Adsorption of fluorescently labeled albumin and lysozyme onto BCP modified PDMS slabs (a) with none remedy (b) after IPA soak and 1 week after O2 plasma. Samples have been lined with protein options for 30–90 minutes. PDMS with PDMS-PEG BCP additive confirmed excessive adsorption, whereas PDMS modified with 1% w/w PDMS-PEG block copolymer exhibited considerably decrease adsorption, close to detection restrict (n = 5). Picture scale bar: 400 μm. (c) We measured adsorption of IgG, BSA, and lysozyme in modified microfluidic units, evaluating the inflow and efflux concentrations after IPA soaking and per week after O2 plasma. PDMS-PEG BCP additive results in considerably lowered adsorption for all proteins. Error bars symbolize the customary deviation with samples measured in triplicate (n = three).

PDMS with no PDMS-PEG BCP adsorbed considerably extra protein than all PDMS with PDMS-PEG-BCP components, confirming that this method led to decreased non-specific adsorption (Fig. 5a). PDMS with zero.125% PDMS-PEG BCP exhibited some protein adsorption. No adsorption was seen for any of the opposite samples. The identical pattern continued following soaking in IPA and O2 plasma remedy (Fig. 5b). PDMS slabs with PDMS-PEG BCP components indicated considerably lowered adsorption as in comparison with additive free PDMS.

To additional quantify protein adsorption in a extra sensible setting for biomicrofluidic system functions, we manufactured microfluidic units from PDMS with or with out PDMS-PEG components. We then launched a protein resolution containing zero.05 mg/mL BSA, lysozyme or IgG into the microchannel (30–90 min), and measured the lack of protein resulting from adsorption on the system by micro-BCA evaluation (Fig. 5c). Gadgets with PDMS-PEG components adsorbed considerably decrease portions of every protein as in comparison with PDMS with no PDMS-PEG (Fig. 5c). Because the BCP focus elevated within the combination, the quantity of adsorbed protein decreased. PDMS-PEG BCP components considerably lowered protein adsorption at concentrations as little as zero.125% (w/w). The usage of just one% PDMS-PEG additive led to 98.9%, 89.four%, and 99.6% decrease adsorption of albumin, lysozyme, and IgG, respectively when in comparison with PDMS with out PDMS-PEG BCP. An additive focus of zero.25% PDMS-PEG led to a ~90% the discount in protein adsorption whereas additionally retaining glorious optical readability.

Capillary-driven microfluidic units with PDMS-PEG BCP components

Having demonstrated the profitable hydrophilization of PDMS with PDMS-PEG BCP components, we investigated the circulate attribute of PDMS with and with out PDMS-PEG BCP components (zero.25% and zero.5%) within the capillary microchannels which have been bonded on the glass substrates. Two linear channels (peak: zero.1 mm, size: 40 mm) with totally different widths (zero.25 mm and zero.5 mm) have been examined for capillary-driven circulate experiments. PDMS with no PDMS-PEG BCP was utilized as a management. All samples have been examined three days after plasma remedy. Liquid was launched into the inlet of the capillary channel and fluid circulate by means of the channel was recorded by a digicam to calculate the experimental circulate charges. Desk three and Fig. S7 present the variation of circulate velocities of liquid utilizing PDMS samples with various quantities of PDMS-PEG BCP. All modified units have been proven to fill by means of regular capillary motion whereas PDMS with out PDMS-PEG BCP did not fill with liquid. We didn’t observe a major distinction in capillary circulate charges by means of the zero.25% and zero.5% PDMS-PEG BCP modified samples. This was in keeping with the WCA outcomes, which confirmed very comparable hydrophilicity (Fig. 2). The benefit of the fabrication approach offered right here is that hydrophilic PDMS microfluidic channels could be obtained with a easy, one-step technique by means of cheap bench-top strategies.

Desk three Capillary-driven circulate of hydrophilic PDMS channels with aqueous options.

We lastly evaluated our outcomes to decide on probably the most most popular PDMS-PEG BCP focus for a given software. Because the biocompatibility and mechanical properties of all PDMS samples with PDMS-PEG BCP components are nearly equivalent with PDMS, we in contrast the totally different samples for his or her transparency, WCA after plasma remedy (t = 45 min) and protein (IgG as a pattern protein) adsorption information. We chosen zero.25% PDMS-PEG BCP focus (WCA = 25.7° ± 2°, transmittance = 99%, discount in IgG adsorption relative to PDMS with no additive = 92.2%) to be one of the best performing composition for functions the place optical readability is of significance, for the reason that transparency of the modified samples decreased all the way down to 73% and under after IPA soaking with BCP concentrations at or above zero.5%.


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