Bioinspired Repellent Pipette Tips with Low Retention Properties Prevent Contamination in Handling Biological Samples

Cross-contamination of biological samples during handling and preparation, is a major issue in laboratory setups, leading to false-positives or false-negatives. Sample carryover residue in pipette tips contributes greatly to this issue. Most pipette tips on the market are manufactured with hydrophobic polymers that are able to repel high surface tension liquids, yet they lack in performance when low surface tension liquids are involved. This presents an obstacle for pipette tips as inaccuracies and loss in precision arise when low surface tension liquids such as viscous oils are pipetted. Here we propose the use of lubricant-infused surface (LIS) technology to achieve omniphobic properties in pipette tips. Using a versatile and simple design, the inner lumen of commercially available pipette tips was coated with a uorosilane (FS) layer using chemical vapor deposition (CVD). We show that after lubricating the tips through simply pipetting up and down a uorinated lubricant, the surface free energy of the tips drastically decreased enabling them to attain low retention properties. Contact angle measurements reveals that the treated pipette tips have enhanced omniphobic properties. The repellent behavior of the lubricant-infused pipette tips against physical adsorption is investigated through pipetting a food coloring dye as well as human blood samples and are compared to the untreated tips. The results show signicantly less amount carryover residue when the lubricant-infused tips are utilized compared to commercially available ones.


Introduction
Fluid carryover in liquid handling devices can lead to experiment failure, measurement inaccuracies and sample loss. It is the major cause of cross-contamination in general scienti c procedures such as bacteriological work, polymerase chain reaction (PCR), and radioimmunoassay [1][2][3][4] . For instance, in PCR ampli cation reactions in a criminal forensic lab, small amounts of DNA contamination could result in amplifying DNA to promote false positive identi cations 5 . In such instances, an underlying cause of contamination could originate from the pipetting of substances with highly viscosities and low surface tensions which can stick to the plastic surface of the pipette, resulting in an improper ejection onto the next test sample 6-8 . Moreover, carryover contamination in pipetting can lead to erroneous volume determination, the need to change tips, limitations to xed pipettes which require to work with open tubes, carbon footprint and costs associated with disposing of single use tips. In order to substantially minimize the effect of carryover, laboratory work areas need separate sets of supplies and equipment, such as pipettes, test-tube holders, and centrifuges [9][10][11] . Single-use tips with lters are usually recommended as the main strategy to prevent amplicon contamination originated form amplicons that accumulate within pipettor 10,12,13 . However, single-use tips are not applicable to automated robotic workstations with their use of xed tips 14 . Techniques have currently been put in place for prevention of cross-contamination, through increasing well-to-well spacing and the prevention of involuntarily z-axes movements from robotic gripper during plate transfer 10,15 . In addition, present literature suggests xed tips treated with robust washing routines are approving of casework samples and serve as a viable and effective alternative to disposable tips 15 . The issue of carryover contamination can be broadened towards other forms of laboratory equipment, such as the syringes and needles used in the removal of PCR products on the robotic arm of automated ampli cation systems 16 . A potential solution would involve surface modi cations to laboratory equipment, namely pipette tips, such that the issue of sample carryover can be minimized.
Most pipette tips on the market are manufactured with hydrophobic polymers that repel high surface tension liquids like water. One of the most commonly used polymers to mold pipette tips is polypropylene due to its hydrophobicity, cost effectiveness, and availability. While hydrophobic and superhydrophobic surfaces are useful for repelling high surface tension liquids, they lack in performance when low surface tension liquids are involved. Thus, this presents an obstacle for pipette tips as inaccuracies and loss in precision arise when low surface tension liquids such as viscous oils are pipetted.
To address this issue, the surface of the tip must be modi ed such that it exhibits low a nity to low surface tension liquids. Such surfaces, referred to as omniphobic, are characterized by their ability to repel all liquids despite their cohesive force strengths 17,18 . A static contact angle of 90° or higher accompanied by a low dynamic angle hysteresis and a low sliding angle of 5° or less are generally characteristic of omniphobic surfaces 19,20 . Omniphobic surfaces can be fabricated using two general techniques: physical and chemical modi cations. Physical modi cation involves roughening the surface using methods such as nano particle deposition, lithographic imprinting and etching [21][22][23] . Chemical modi cation lies on the basis of decreasing the free energy of the surface of interest 1,24,25 . Several surface modi cation techniques that rely on altering the surface chemistry are known, some of the most prevalent include the use of uorocarbon compounds or organosilanes as surface coatings 21,26 .
The growing demand for pipette tips with minimal sample surface adherence has led to the development of low retention pipette tips. Low retention pipette tips exhibit omniphobic surface properties thereby leading to minimal sample loss. One example involves using unique molding processes to incorporate uoropolymer molecules into the surface of the pipette tips to achieve hydrophobic properties 27 .
Texturing the pipette tips is another technique used by researchers to hydrophobically coat the pipette tips 28,29 . These methods, however, present negative aspects such as increased manufacturing complexity, high cost and lower effectiveness as compared to surface coating.
Here, we propose the use of CVD treatment with a simple apparatus designed to achieve omniophobic properties in pipette tips. CVD treatment of a uorosilane can drastically reduce the surface free energy of the tip enabling it to attain low retention properties. The uorosilanized tips are then lubricated with a uorinated lubricant to generate lubricant infused surfaces (LIS) with omniphobic properties. Some of the bene ts of this method include process simplicity, lower processing time, high e ciency and reduced cost.

Materials
Trichloro (1H, 1H, 2H, 2H-per uorooctyl) silane (TPFS), per uoroperhydrophenanthrene (PFPP) (Sigma, Oakville, Canada), pipette tips composed of polypropylene (Diamed Lab Supplies Inc., Mississauga, Canada), red food coloring dye (A Preema Quality Product, Ingredients: Sodium Chloride and E122 Carmosine), and tween 20 (Sigma, Oakville, Canada) were used as received. Citrated human blood was generated from blood samples collected from healthy donors. All procedures were approved by the McMaster University Research Ethics Board. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all the donors.

Methods
Pipette tips were placed onto a cylindrical shaped apparatus acting as a holder device. Holder apparatus was placed in an oxygen plasma cleaner (Harrick Plasma Cleaner, PDC-002, 230 V) and exposed to highpressure oxygen plasma for 2 minutes for surface functionalization to enable reaction with TPFS. Upon oxygen plasma-treatment and removal from the plasma cleaner, the holder apparatus with pipette tips and petri dish with control tips samples were placed in a desiccator connected to a vacuum pump. 200 µL of TPFS was pipetted onto a glass slide on a separate petri dish located across from the holder apparatus. 100 µL of TPFS was also pipetted on glass slide located inside the holder apparatus ( Fig. 1).
The desiccator was vacuumed at a pressure of −0.08 MPa, therefore initiating the CVD treatment. The silanization reaction took place over a period of 2.5 hours at room temperature. Upon completion of CVD, pipette tips were removed from the holder device and heat treated at 60°C overnight. In order to infuse lubricant in the uorosilanized pipette tips, the PFPP lubricant was simply pipetted in and out.
FT-IR (Bruker, Karlsruhe, Germany) was used to assess the surface chemical composition of the pipette tips before and after the treatment. During FT-IR measurements, the air was considered the background, and the other spectra of the uorosilanized and untreated tip surfaces were normalized based on this baseline. Contact angles of the uorosilanized and untreated were measured using 5 µL droplets of water, tween 20, and isopropanol. Water sessile drop contact angle measurements were performed at room temperature before and after each modi cation step using a Future Digital Scienti c OCA20 goniometer (Garden City, NY), which was calibrated prior to each measurement.
The blood cells and proteins interactions with lubricant-infused and untreated pipette tips were assessed with recalci ed blood. Citrated human blood was recalci ed by CaCl 2 diluted in HEPES at a concentration of 12.5 mM. For scanning electron microscopic (SEM) imaging (JSM-7000 F), after pipetting up and down the recalci ed blood, the tips were cut and xed using 2% glutaraldehyde diluted in PBS. The tips were then incubated in 1% osmium tetroxide in 0.1 m sodium cacodylate buffer for an hour, followed by dehydration via a graded series of ethanol and critical point drying by Leica EM CPD300 dryer (Leica Mikrosysteme GmbH, Wien, Austria) using liquid CO 2 ush. Before imaging, the samples were gold sputtered (Polaron Model E5100 sputter coater, Watford, Hertfordshire).
In the dye experiments, 10X serial dilutions of the red dye were performed in a well-plate to assess carryover of treated tips. The initial concentration of the dye was 1 mg mL −1 . The dye was serially diluted in water containing 0.05% tween 20 using lubricant infused and untreated tips for comparison. For the purpose of the carryover evaluation, the pipette tips during the serial dilutions were not changed and the same pipette tip was implemented throughout all the dilution steps. The well-plate was analyzed using a plate reader (Tecan In nite M1000) to nd the absorbance values.

Results And Discussion
In order to uniformly coat the inner surface of the pipette tips composed of polypropylene (Diamed Lab Supplies Inc.), they were placed onto a cylinder-shaped apparatus which acted as a holding device. The apparatus with tips were oxygen plasma treated and silanized with trichloro (1H, 1H, 2H, 2Hper uorooctyl) silane (TPFS) using the CVD method. This allows the hydrophilic terminal (trichlorosilane) of the uorosilane molecule to bind to the plasma induced hydroxyls on the surface of the tips, resulting in self-assembled monolayers (SAMs) of uorosilane (FS) with an umbrella-shaped structure in a way that the uorine terminals are exposed on the surface (Fig. 1). The uorinated tips were then lubricated by simply pipetting in and out a uorinated lubricant called per uoroperhydrophenanthrene (PFPP).
To analyze the changes in the chemical composition of the pipette tip surface after oxygen plasma treatment and after CVD modi cation, fourier-transform infrared spectroscopy (FT-IR) was performed on 1 mL uorosilanized and untreated pipette tips (Fig. 2a). The spectra of both uorosilanized and untreated tips had a number of peaks in the absorption band between 800 to 1200 cm −1 , speci cally at 850 cm −1 , 1000 cm −1 , and 1200 cm −1 vibrations that are characteristic for isotactic polypropylene. These absorption bands can be interpreted as vibrations of C-H, CH 2 and CH 3 groups in the polymer chain 30 . In the uorosilanized tips, however, a broad absorbance band is present around the 3700-3200 cm −1 . This is believed to correspond to the Si-OH group bonds formed as a result of the hydrolysis of TPFS molecules.
It should be mentioned that 3700-3200 cm −1 is the range of absorbance bands that commonly corresponds to alcohols and phenols, including bonds to silicone compounds 30 . This pattern is notably absent when looking at the untreated tip sample spectra, which produces a at line over the 4000-3000 cm −1 region (Fig. 2a).
To examine the relative hydrophobicity/hydrophilicity of the treated and untreated pipette tips, contact angle measurements were performed using a 5 µL droplet of deionized water. The static contact angle measurements of the untreated tips, uorosilanized tips, and lubricant infused tips are shown in Fig. 2b. Untreated tips demonstrated a lower contact angle (θst= 75 ± 11°) implying low levels of hydrophobicity.
It is worth noting that the standard deviation value of the untreated samples is relatively high due to the conditions of the untreated tip surface. Upon CVD treatment and before lubricant addition, the contact angle increased to 89 ± 3°. Finally, after addition of PFPP lubricant to the uorosilanized tips, contact angles of water were demonstrated to increase to 101.7 ± 7°.
Due to the surface curvature of the pipette tip, measuring the contact angle of a liquid droplet on the tip using a tensiometer presents complications and might result in slight inaccuracy. As such, the contact angle of water, tween 20, and isopropanol as a low surface tension liquid were measured on treated and standard pipette tips using capillary rise method. Capillary rise can be utilized to compute the contact angle of the rising/dropping liquid using hydrostatics principles. Due to the close resemblance of the tips to a truncated cone shape, the capillary rise equation was modi ed to account for such capillary shape (equation 1): where θ is the contact angle, ρ is the density of the liquid, g is the gravitational constant, h is the rise/drop height, R is the diameter of tube at the three-phase region, γ is the surface tension of the liquid, and β is the conical angle of tip.
As such, the contact angles of liquids with various surface tensions on the uorosilanized and untreated tips were acquired to test the effect of surface modi cation on omniphobic properties of the tips (Fig. 2c). The contact angles of all three liquids were higher on the uorosilanized tips compared to untreated tips. This is especially evident for the lower surface tension liquids such as the tween 20 and isopropanol. The increase in the contact angles of the treated samples highlight the higher surface repellency induced by the treatment. The contact angle of tween 20 was 30° higher on the uorosilanized surface as compared to the standard while that of isopropanol was 28° higher. Since the tips exhibited hydrophobic properties even prior to surface treatment due to the properties of the material used to manufacture the tips, there was a smaller difference in contact angle of water on the treated surface with 6.9° increase using the capillary rise method and 40° increase using the optical tensiometer. Notable, the hydrophobic nature of the intact pipette tips was not fully appreciated in Fig. 2b which could be due to the fact that the surface properties of the tips somewhat changed when the tips were cut and attened for the purpose of the contact angle measurement.
In order to study the e cacy of our surface medication in preventing the physical (non-speci c) adsorption of biomolecules and reagents, we rst performed a visual test through pipetting in and out a food coloring red dye diluted in water containing 0.05% tween 20. As it can be seen in Fig. 3a, the untreated 200 μL pipette tip showed a great amount of dye adsorption onto the tip, whereas the lubricant infused tip maintained signi cantly lower amount of dye mostly at the bottom end of the tip after pipetting out the dye. In the next experiment, we used lubricant infused tips at different sizes of 10 μL, 200 μL, and 1 mL to pipette in and out recalci ed citrated human blood. As it is demonstrated in Fig. 3b, the lubricant infused tips in all sizes could effectively suppress blood adhesion and clot formation inside the tip in comparison with the untreated tips. SEM images in Fig. 3c also exhibits the presence of blood cells and clot on the surface of the untreated tips, while the lubricant infused modi cation signi cantly reduced the cell attachment and clot formation.
To further quantify the effects of lubricant infused technology on the functionality of the pipette tips, 10fold serial dilutions of 1 mg mL −1 dye were performed in water containing 0.05% tween 20. The same pipette tip was used to perform all the dilution steps to better illustrate the effect of dye residue inside the tip during the serial dilutions. Using absorbance measurements (at the wavelength of 512 nm), we quanti ed the results obtained from lubricant infused and untreated pipette tips in two sizes of 10 µL and 200 µL shown in Fig. 4a and b, respectively. The results of the dilutions indicate that the untreated tips result in have signi cantly higher carryover residue; as a result, the dilutions did not occur e ciently. Thus, the more amount of dye that can be seen at lower concentrations could be due to the dye residue that had remained onto the tip surface from the initial high concentrations and was carried to the low concentrations.
As can be seen from the Fig. 4a and b, the lubricant infused tips show a steeper decrease in absorbance per dilution number followed by a sooner levelling off to the diluent's absorbance value (with zero concentration of the dye) that is indicated by dashed line in Fig. 4a. On the other hand, the untreated tips reveal higher absorbance levels even for higher dilution numbers which is a result of the sample adhering to the tip's wall even after performing several dilutions. In addition, there is signi cantly higher variation observed with untreated tips compared to treated ones as indicated by large error bars shown in Fig. 4a and b. In Fig. 4c, the colour difference in the well-plate after performing the serial dilutions via the lubricant infused tips and untreated tips could be observed.

Conclusions
Sample adherence to tip wall during pipetting activities introduces many problems downsizing experimental accuracy. Current techniques to reduce this effect are expensive and require modi cations to the plastic mix used for manufacturing the pipette tips. This work proposes a simple surface modi cation technique to induce liquid/protein repellency on the inner and outer walls of the pipette tips. The proposed lubricant infused tips with omniphobic properties are able to repel low and high surface tension liquids. The coating will reduce sample carryover and adherence to the tip wall. This technique can thus be used to minimize cross-contamination of experiments involving highly vicious surfaces such as clotting blood and increase e ciency by decreasing sample loss due to carry over adherence. Declarations S.K and D.A.M, and inputs from M.R. and T.D. All authors have read and approved the nal version of the paper.

Competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Data availability
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.  Schematic illustration of the treatment procedure along with the chemical structures of the treated tips at each step of the modi cation. Pipette tips in specially designed holder apparatus were CVD treated with TPFS followed by heat treatment at 60 ℃ overnight. The tips were subsequently lubricated via pipetting in and out PFPP lube.