Reports Reports Interference with spectrophotometric analysis of nucleic acids and proteins by leaching of chemicals from plastic tubes L. Kevin Lewis, Michael H. Robson, Yelena Vecherkina, Chang Ji, and Gary W. Beall Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA

BioTechniques 48:297-302 (April 2010) doi 10.2144/000113387 Keywords: ; polypropylene; plastic additives; microcentrifuge tube; leachate

Absorbance spectroscopy is routinely used to monitor the concentrations of nucleic acids and proteins within solu- tions and to assess changes in their structure caused by interaction with chemicals or other biomolecules. Biological samples used for such analyses are manipulated and stored in small microcentrifuge tubes (microtubes) composed of polypropylene plus several plastic additives. Here we demonstrate that normal handling of laboratory microtubes causes leaching of -absorbing chemicals into biological samples that interfere with spectrophotometric measure- ments. The leached chromophores absorbed UV light strongly at 220 and 260 nm, which are the wavelengths nor- mally used to detect and quantitate proteins and DNA. Some common laboratory techniques, including sonication and PCR, were particularly effective inducers of leaching. The magnitude of the increase in absorbance was depen- dent upon both time and heat history, with greatest induction after tubes were warmed to temperatures at or above 37°C. revealed that aqueous solutions stored in plastic microtubes accumulate a complex mixture of leached chemicals with molecular masses of 200–1400 Da. Leaching was ubiquitous among commercially available brands of microtubes, indicating a persistent source of error in biomolecule detection and concentration measurements.

as chemical antioxidants, mold release employed. Polypropylene, like other Introduction agents, biocides, and UV-light stabilizers commercial plastics, contains multiple Detection and quantitation of nucleic (6,7). Plastic materials may also contain additives that protect and preserve the acids within biological samples are other low–molecular weight chemicals integrity of the polymer material (7,12). routinely performed by monitoring such as residual unreacted monomers and Tubes molded from this plastic have absorbances of solutions at 260 nm. polymer degradation products. Several proven invaluable for biological research Absorbance spectrophotometry is also a past studies have demonstrated that because of their useful mechanical valuable tool for analysis of alterations in additives and other chemicals can leach properties, resistance to extreme tempera- the secondary structure of DNA, RNA, from plastics into the environment (8–13). tures, and low chemical reactivity. and related molecules such as poly(ADP- A diffusable monomer component within ribose) (1,2). This latter assessment takes polycarbonate, bisphenol A (BPA), has advantage of the well-known hyperchro- received much attention in recent years Materials and methods micity of DNA and RNA, resulting because of its widespread distribution UV light absorbance spectroscopy from intrinsic differences in absorbance and its ability to act as an endocrine UV absorbance measurements utilized among nucleotides and single-stranded disruptor in animals (8,9). Concern a SmartSpec 3000 spectrophotometer and double-stranded species. In addition, has also been raised about release of (Bio-Rad, Hercules, CA, USA) with absorbance measurements at 220 nm formaldehyde, antimony, phthalates and the scan smoothing function activated. and 280 nm are frequently employed to various polymer breakdown products Leachate absorbance experiments detect and quantitate proteins, while other from drinking bottles and other plastic described in Figures 1–2 were performed wavelengths within this range are used to containers (10–13). using VWR polypropylene microtubes analyze structural changes induced within Biochemistry researchers use small, (Cat. no. 20170–038; Schertz, TX, USA) DNA and RNA upon binding of biomol- 1.5-mL polypropylene microcentrifuge unless specified otherwise. DNA oligo- ecules or exogenous chemicals (1,3–5). tubes (microtubes) during purification, nucleotide Pvu4a (AAATGAGTCAC- Plastic containers used for the storage handling, and storage of nucleic acids, CCAGATCTAAATAA) was obtained and manipulation of biological samples are proteins, and other biological samples. from BioServe Biotechnologies (Laurel, constructed of complex polymer materials Specialty tubes, such as the thin-walled MD, USA) and analyzed using 77 µg/mL containing multiple chemical additives. 0.2-mL polypropylene microtubes used solutions. DNA concentration calculations These additives include supplements such for PCR experiments, are also frequently were performed using the relationship that

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Figure 1. Absorbance characteristics of chemicals leaching from common laboratory microtubes. (A) Brief heating (30 min, 100°C) released UV-absorbing chemicals into water that produced either type 1 or type 2 spectra. Most commercially available tubes are type 1. Microtubes: type 1, VWR, Cat. no. 20170–038; type 2, Ambion, Cat. no. 12400. (B) Temperature- and time-dependence of 218-nm peak formation in the most common tube type. Error bars indicate s d . (C) Alteration of absorbance spectra of single-stranded oligonucleotide DNA (25 nucleotides) after incubation for 30 min at 95°C. –Δ, no heat; +Δ, heated sample.

an A260 of 1.0 corresponds to a concen- Applied Biosystems (Cat. no. AM12225), 50 µL/min was used as the push solvent. tration of 33 µg/mL for single-stranded Nalgene (Genunc, Cat. no. #250865; Lima, The mass spectrometer experimental condi- DNA oligonucleotides (1). Chloroform, OH, USA), ISC BioExpress (GeneMate, tions were as follows: sheath gas flow rate, DMSO, and methanol were obtained from Cat. no. C-3257–1), and VWR brand tubes 75 (arbitrary units); aux gas flow rate, 20; Sigma-Aldrich (St. Louis, MO, USA). PCR (Cat. no. 20170–010). and capillary temperature, 200°C. Spray was simulated using a 2720 Thermal Cycler voltages were 5 kV (positive ion mode) and from Applied Biosystems (Foster City, CA, GC–mass spectrometry 3.5 kV (negative ion mode). USA). Sonication was performed using a An Agilent Technologies (Santa Clara, Vibracell VC130 sonicator (Sonics & CA, USA) model 6890N gas chromato- Materials, Inc., Newtown, CT, USA) set graph (GC) equipped with a model 5973N Results and discussion at an amplitude of 30. Standard micro- mass selective detector was utilized for As part of a recent spectrophotometric centrifuge tubes tested in these studies analysis of the leachate for compounds study of the efficiency of sedimentation included the following brands and catalog with molecular masses up to 200. Analytes of DNA and clay particles by centrifu- numbers: VWR (Cat. no. 20170–038), were separated using an Agilent Technol- gation (14), we observed that prolonged Ambion/Applied Biosystems (Cat. nos. ogies HP-5MS 30 mm × 0.25 mm capillary high-speed centrifugation and concom- 12400 and 12300), Sarstedt (Fisher, column with a 5% crosslinked phenylm- itant warming of plastic microtubes caused Cat. no. 50809206; Newton, NC, ethylsiloxane stationary phase. The oven an unexpected increase in UV absorbance USA), Axygen MCT-175-A (VWR Cat. temperature was held initially at 45°C at 260 nm. To investigate the phenomenon no. 10011–718), Intermountain Scien- for 6 min and then ramped to 200°C at more comprehensively, absorbances of tific Corporation (Cat. no. C-3261–3; 8°C/min. The split ratio was 20:1. several commercially available brands of Kaysville, UT, USA), Vangard (Cat. no. standard microtubes containing 1 mL CN065S-GT; Vangard International, Electrospray ionization deionized water were analyzed before Inc., Neptune, NJ, USA), and Fisherbrand mass spectrometry and after heating the tubes for 30 min in (Fisher, Cat. no. 02–681–376). Two tubes Positive-ion and negative-ion electrospray a 100°C heating block. New absorbance recently advertised to contain fewer mold ionization experiments were performed peaks appeared at approximately 220 and release agents (Axygen MCT-150-C; VWR using the conventional electrospray 260 nm upon heating, producing two 10011–700) or plasticizers, slip agents, and ionization source of an LCQ Classic consistent patterns (Figure 1A). Most tubes biocides (Cat. no. 022364111; Eppendorf, (Thermo-Fisher, Waltham, MA, USA) displayed the type 1 spectrum, with a large Hauppauge, NY, USA) were also tested. operating at m/z range of 200–2000. peak near 220 nm (218 nm maximum) PCR tubes (0.2-mL, thin-walled) tested Sample solutions were introduced into the and smaller peak near 260 nm (259 nm included the following brands: Labcon electrospray ionization source using a 5-µL maximum). In a minority of tubes, the (VWR, Cat. no. 21070–012), Ambion/ sample loop. Methanol with a flow rate of peak near 260 nm was dominant. No other

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Figure 2. Impact of common biochemistry laboratory procedures and solvents on leachate formation within polypropylene microtubes. (A) Spectra observed for leachates produced, without heating, after 30 min in common biochemistry solvents chloroform (CHCl3), methanol (MeOH), and DMSO. (B) Seepage of UV-absorbing materials from thin-walled 0.2-mL tubes into water is substantial after 30 cycles of PCR (top panel). Modest leaching was also observed after centrifugation of standard 1.5-mL tubes for 30 min at 18,000× g or mild sonication for 5 min. Control, untreated solution. PCR microtubes: type 1, Ambion, Cat. no. AM12225; type 2, Nalgene Genunc, Cat. no. 250865. (C) Electrospray mass spectra of water leachate (30 min, 95°C) from type 1 (VWR, Cat. no. 20170–038) microtubes. absorbance peaks were observed within the presence of double bonds within amino but strong at room temperature (i.e., wavelength range of 200–600 nm in any acid carbonyl groups. Most proteins also without heating) within tubes containing of the tubes tested. absorb light at 280 nm, with peak height the common biochemistry solvents A representative tube from the most at 280 nm dependent primarily upon the chloroform, methanol, or DMSO (Figure common group—type 1—was used for fraction of tryptophan and tyrosine amino 2A). Incubation with chloroform—often subsequent studies of leaching from acids within the protein. To examine used for DNA extractions—produced the standard 1.5-mL tubes except where noted how biomolecule absorbance spectra are highest leachate response of all solvents otherwise. The magnitude of leachate shifted by heating in microtubes, aqueous tested (absorbance > 2.0). Peak patterns peak absorbance could be substantial solutions containing a single-stranded displayed by the organic solvent leachates (up to 1.0–2.0 absorbance units) and was DNA oligonucleotide (25 nucleotides were typically not identical to those temperature-dependent (Figure 1B, top in length) were scanned before and after produced in aqueous solutions, which is panel). Modest chromophore leaching heating for 30 min. As shown in Figure likely a reflection of differential solubilities was detectable after 30 min at 37°C, 1C, heating caused a substantial increase in of the leachate chemicals and changes in the temperature used for most enzyme- absorbance at 260 nm in type 1 tubes. This solvation effects on absorbance. catalyzed reactions in biochemistry, but increase, from 0.31 to 0.48, is equivalent to The impact of common laboratory seepage was strongest at higher tempera- an apparent change in DNA concentration methods on chromophore leaching was also tures (70°C and 95°C). These high temper- from 10.2 to 15.8 µg/mL, an overestimate evaluated. PCR, one of the most frequently atures are often employed to heat-inactivate of 55%. In the type 2 tubes analyzed in employed techniques in biochemistry, uses enzymes, denature DNA, and perform Figure 1C, a larger increase in absorbance thin-walled polypropylene tubes manufac- other reactions such as PCR that involve was observed (0.24 to 0.96), suggesting a tured specifically for this procedure. thermostable enzymes (described below). change from 7.9 to 31.7 µg/mL, resulting Several brands of PCR tubes containing The effect was time-dependent and rapid, in an overestimate of 300%. No increase 150 µL water were all found to leach being readily observable <5 min after in absorbance occurred when heating light-absorbing materials after exposure exposure to elevated temperatures (Figure was performed using a glass test tube as to a standard 30-cycle program (30 s at 1B, bottom panel). control. 95°C, 30 s at 60°C, and 2 min at 72°C) Nucleic acids absorb light strongly Most plastic additives and their degra- on a PCR thermocycler (Figure 2B, top at 260 nm and absorbance at this dation products are small- and medium- panel). Absolute absorbances achieved were wavelength is commonly employed to sized organic molecules and therefore may strong but variable, depending upon the detect and quantitate DNA (1). Proteins seep most efficiently into organic solvents. brand tested, and could be classified into absorb UV light at 220 nm due to the We observed that leaching was variable two patterns as before. Other laboratory

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procedures, such as centrifugation in a 100°C), we measured initial absorbances did not identify any components with microcentrifuge at 18,000× g for 30 min and then performed DNA precipitations masses <200 Da when the water leachate or mild sonication for 5 min (Figure 2B, according to a standard protocol utilizing was dried and resuspended in either bottom panels), also stimulated leaching. sodium acetate and ethanol (1). The A220 acetone or diethyl ether. However, after Increasing the centrifugation or sonication of the initial leachate solutions was 0.376 direct extraction of the water leachate into times produced higher absorbances and also ± 0.015 (± sd) and became 0.010 ± 0.009 diethyl ether, a major component with a greater warming of the tubes, suggesting after the precipitated material was resus- mass of 134 Da was detected and predicted that the increase in temperature is the pended. Using the same procedure, double- to correspond to 2,4-dimethylbenzal- primary leaching stimulus. stranded plasmid DNA was precipitated dehyde or 2-ethylbenzaldehyde. Biochemists routinely visualize DNA with 90% efficiency, indicating that the In this work, we have demonstrated that and RNA in gels by staining with the method used could effectively precipitate plastic microtubes used in biochemistry fluorophore ethidium bromide, and DNA DNA molecules. These results demonstrate research release chemicals into aqueous is quantitated in solutions using fluoro- that the light-absorbing leachate materials solution that absorb light at DNA and phores such as Hoescht 33258. We did not do not readily co-precipitate with DNA. protein absorbance peak maxima during detect interference in ethidium bromide This finding is not unexpected becausei ( ) normal use. Leaching was promoted by staining or Hoescht 33258–based DNA the mass spectrometry data revealed that procedures that cause warming of the quantitation assays by leachates (data not chemicals leached from microtubes have tubes such as high-speed centrifugation, shown), possibly because of the specificity low molecular weights (<1400 Da) and sonication, and incubation at tempera- of these chemicals for DNA and because (ii) small molecules, including very small tures ≥37°C. their emissions are at visible wavelengths DNAs, are not precipitated efficiently out Commercial polymers typically contain (above 400 nm) and leachate chemicals do of aqueous solutions. several different additives, many of which not have strong interactions with light at The chemical composition of the water contain double bonds and aromatic groups these wavelengths. leachates was investigated using electro- that cause them to absorb UV light (6,7). We also performed experiments to test spray mass spectrometry, a non-fragmenting Many additives, such as antioxidants and whether leachate chemicals are coprecipi- technique with an approximate detection UV protectants, act as sacrificial defenders tated during precipitation of DNA with range of 200–2000 Da. This analysis that become chemically altered and/or ethanol, which is a routine procedure in revealed that water leachates are hetero- degraded during polymer heating and many molecular biology labs. Using type geneous mixtures of small molecules with processing and also over time as the plastic 1 microtubes containing high amounts masses between 200 and 1400 Da (Figure ages (6,7,15,16). The large size heteroge- of leachate produced by heating using 2C). Use of GC-mass spectrometry, which neity of the materials diffusing from the our standard method (H2O, 30 min, detects the presence of smaller molecules, plastic microtubes suggests that leachates

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are composed of multiple additives and may interfere with functional assays for a and base excision. Proc. Natl. Acad. Sci. USA their breakdown products. In widely few specific biomolecules, but our obser- 105:70-75. available polymers such as polypropylene, vations suggest that a more widespread 5. Ghasemi, J., S. Ahmadi, A.I. Ahmad, and the processing, heating, and oxidation S. Ghobadi. 2008. Spectroscopic character- problem exists. ization of thiazole orange-3 DNA interaction. histories of different preparations will vary, The ubiquitous nature of leaching Appl. Biochem. Biotechnol. 149:9-22. as will the effects of aging. It is likely that among the different brands of micro- 6. Pfaendner, R. 2006. How will additives shape the variations in UV absorbance spectra of tubes tested indicates that this contam- the future of plastics? Polymer Degrad. Stabil. the different tubes (Figure 1A and Figure ination is a persistent source of error in 91:2249-2256. 2B) are a reflection of this variability in molecular biology experiments utilizing 7. Zweifel, H. 2009. Plastics Additives Handbook, polymer compounding methods and UV spectroscopy. The magnitude of such 6th ed. Hanser Publications, Munich. stress history, as well as in the types errors can be substantial, since leachates 8. Vandenberg, L.N., M.V. Maffini, C. Sonnen- schein, B.S. Rubin, and A.M. Soto. 2009. and quantities of additives used. Some typically exhibited A260 readings between companies have recently begun manufac- Bisphenol-A and the great divide: a review 0.2 and 2.0 absorbance units. Mitigation of controversies in the field of endocrine turing microtubes that contain a reduced of the problem in the future will require disruption. Endocr. Rev. 30:75-95. number of chemical additives. The precise use of plastics that retain the positive 9. Oehlmann, J., M. 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Indoor residential magnitude of leaching observed using our that some manufacturers (see “Materials standard assay (H O, 30 min, 100°C; data chemical emissions as risk factors for respi- 2 and methods” section) have announced ratory and allergic effects in children: a review. not shown) was reduced in two such tubes new polypropylene-based tubes that Indoor Air 17:259-277. (see “Materials and methods” section). contain fewer additives, though the impact 11 . Shotyk, W., M. Krachler, and B. Biochemistry labs frequently employ of such changes on the beneficial charac- Chen. 2006. Contamination of Canadian and plastic 96-well microtiter plates, usually teristics and lifespan of the tubes is not yet European bottled waters with antimony from made from polystyrene, to perform enzyme clear. PET containers. J. Environ. Monit. 8:288- assays and other high-throughput proce- 292. dures. In tests using polystyrene 96-well 1 2 . Malo, J.L., A. Cartier, L. Pineault, plates (Cat. no. 353075; BD Biosciences, Acknowledgments M. Dugas, and A. Desjardins. 1994. Occupa- San Jose, CA, USA) and our standard tional asthma due to heated polypropylene. We thank Mehdi Moini for expert Eur. Respir. J. 7:415-417. assay (H2O, 30 min, 100°C), we did not technical analysis using electrospray mass 1 3 . Heudorf, U., V. Mersch-Sundermann, detect increased absorbance at either 220 spectrometry. This work was supported by and J. Angerer. 2007. Phthalates: toxicology or 260 nm. This suggests that polystyrene- the National Institutes of Health (NIH; and exposure. Int. J. Hyg. Environ. Health based materials used in biochemistry labs grant no. 1R15AG028520-01A1) and the 210:623-634. may be different from polypropylene in Welch Foundation (grant no. AI 0045). 1 4 . Beall, G.W., D.S. Sowersby, R.D. this regard. However, such investigations Roberts, M.H. Robson, and L.K. Lewis. 2009. This paper is subject to the NIH Public Analysis of oligonucleotide DNA binding are complicated by the fact that many Access Policy. microtiter dishes contain special surface and sedimentation properties of montmoril- coatings for tissue culture that may alter lonite clay using light spectroscopy. Biomacromolecules 10:105-112. leachate responses; therefore, further study Competing interests 1 5 . Marcato, B., S. Guerra, M. Vianello, is needed. The authors declare no competing and S. Scalia. 2003. Migration of antioxidant Biochemistry and molecular biology interests. additives from various polyolefinic plastics labs routinely use A260 measurements to into oleaginous vehicles. Int. J. Pharm. quantitate DNA and A220/A 280 readings 257:217-225.. for detection of proteins. UV absorbance References 1 6 . Begley, T.H., J. Brandsch, W. Limm, H. Siebert, and O. Piringer. 2008. Diffusion is also monitored to analyze changes in 1. Sambrook, J. and S.W. 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N’soukpoé-Kossi, D. plasticware. Science 322:917. stimulates the release of UV-absorbing Charbonneau, C.M. Weinert, L. Kreplak, polypropylene leachates. McDonald et and H.-A. Tajmir-Riahi. 2009. Structural Received 21 December 2009; accepted 26 al. reported that additives leached from analysis of DNA complexation with cationic February 2010. lipids. Nucleic Acids Res. 37:849-857. some commercially available polypropylene 4. Minetti, C.A., D.P. Remeta, and K.J. Address correspondence to L. Kevin Lewis, microtubes can inhibit the enzyme human Breslauer. 2008. A continuous hyperchro- Chemistry and Biochemistry, Texas State monoamine oxidase-B (hMAO-B) (17). micity assay to characterize the kinetics and University, 601 University Drive, San Marcos, Their results demonstrate that leachates thermodynamics of DNA lesion recognition TX 78666, USA. e-mail: [email protected] Vol. 48 | No. 4 | 2010 302 www.BioTechniques.com