Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate)

Bacillus pumilus SAFR‐032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H₂O₂, desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR‐032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa‐FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant‐resistant B. pumilus SAFR‐032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa‐FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase‐K, lysozyme, mutanolysin and Triton X‐100 facilitated efficient spore detection by Alexa‐FISH microscopy. Neither of the Alexa‐probes tested gave rise to considerable levels of Lucite‐ or solvent‐associated background autofluorescence, demonstrating the immense potential of Alexa‐FISH for rapid quantification of encapsulated B. pumilus SAFR‐032 spores released from poly(methylmethacrylate).     

Recovery of bacillus spore contaminants from rough surfaces: a challenge to space mission cleanliness control

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference with life detection experiments. Therefore, space agencies measure the cultivable spore load ("bioburden") of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus spores from Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl-Eichinger, Appl. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency), was then compared to the current European Space Agency (ESA) standard wipe assay in sampling four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis). Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research.     

HYDROXYPROPYL METHACRYLATE, A NEW WATER-MISCIBLE EMBEDDING MEDIUM FOR ELECTRON MICROSCOPY

Aldehyde-fixed rat tissues were variously dehydrated and impregnated in water-miscible 2-hydroxypropyl methacrylate (HPMA) containing 3 to 20 per cent water and 0.1 per cent α,α-azobisisobutyronitrile as catalyst for subsequent polymerization with ultraviolet light. Heat polymerization was also effective. Blocks of embedded tissue readily gave ultrathin sections, which required staining by uranyl acetate and/or lead stains to give adequate contrast for electron microscopy. The ultrastructure of pancreas, kidney, muscle, and intestine was well preserved by aldehyde fixation alone. Use of postfixation in osmium tetroxide or direct osmium tetroxide fixation was unsatisfactory. The fine structure of aldehyde-fixed liver from fasted rats was well preserved, whereas that from normal rats showed considerable disorganization and collapse, apparently because of extraction of glycogen during the embedding procedure. Enzymatic extraction of proteins by pepsin and of ribonucleic acid by ribonuclease after either formaldehyde or glutaraldehyde fixation was rapidly effected by direct treatment of ultrathin sections with solutions of the enzymes. In contrast, no digestion of chromatin by deoxyribonuclease could be detected. In spite of this present limitation, HPMA appears to have several advantages over earlier water-miscible embedding media for electron microscopy and to be particularly suitable for ultrastructural cytochemistry.     

The use of a model system to compare the efficiency of ultrasound and agitation in the recovery of Bacillus subtilis spores from polymer surfaces

The effectiveness of ultrasound and mechanical agitation for the recovery of Bacillus subtilis spores from pre-inoculated glass, polystyrene, polypropylene, polyethylene and polycarbonate surfaces was tested in the presence and absence of surfactant. Recovery from polymers was improved by the use of surfactant. Simple agitation, as frequently used in bioburden determination, is shown to be a poor method for the recovery of spores from hard surfaces. The use of ultrasound achieves recoveries over 90% only with glass, recoveries from polymers ranging from 40%–75%. Recoveries from polypropylene and polycarbonate showed poor reproducibilities. Agitation with Ballotini beads improved the reproducibility of recoveries from polymers and also in the majority of cases improved recovery levels.     

Assessment of bioburden on human and animal tissues: Part 2—Results of testing of human tissue and qualification of a composite sample for routine bioburden determination

A quantitative method was developed and validated to assess bioburden on tissue from human donors and to compare bioburden determination results to swab culture results from the same donor. An initial study with allograft tissue from 101 donors showed a wide range of bioburden levels; values from no colony-forming units (CFU) detected to?>28,000?CFU were observed. Tissues from donors that had swab cultures negative for objectionable microorganisms generally had lower bioburden than tissues from donors where objectionable microorganisms were recovered by swab culturing. In a follow-up study with 1,445 donors, a wide range of bioburden levels was again observed on tissues from donors that were swab culture negative for objectionable microorganisms. Tissues from 885 (61%) of these donors had no recoverable bioburden (<2?CFU). Importantly, tissues from 560 (39%) of the donors had recoverable bioburden which ranged from 1 to?>24,000?CFU. Identification of bioburden isolates showed a diversity of genera and species. In compliance with the recent revision of the American Association of Tissue Banks K2.210 Standard, the quantitative bioburden determination method was validated with a composite tissue sample that contains bone and soft tissue sections tested together in one extraction vessel. A recovery efficiency of 68% was validated and the composite sample was shown to be representative of all of the tissues recovered from a donor. The use of the composite sample in conjunction with the quantitative bioburden determination method will facilitate an accurate assessment of the numbers and types of contaminating microorganisms on allografts prior to disinfection/sterilization. This information will ensure that disinfection/sterilization processes are properly validated and the capability of the overall allograft process is understood on a donor by donor basis.     

Assessment of bioburden on human and animal tissues: Part 1—Results of method development and validation studies

Recovered human and animal tissues are used extensively in surgery for wound repair and reconstruction. In preparation for the validation of chemical disinfection and radiation sterilization processes, studies were performed on the development and validation of quantitative bioburden recovery methods for human bone and soft tissue and also for porcine dermis. The use of a swab-based method was not considered due to the known poor efficiency of recovery for this technique. The “exhaustive extraction” and “inoculated product” approaches to validation of a bioburden recovery efficiency factor have inherent strengths and weaknesses; in this study, tissues were inoculated and also subjected to a series of extractions to determine if/when “exhaustion” occurred. Femoral and tibial shaft rings, iliac crest wedges, sections of Achilles tendon, a soft tissue composite sample, and porcine dermis, were inoculated at several sites with Bacillus atrophaeus spores, and then subjected to either shaking by hand, mechanical shaking, or sonication plus mechanical shaking. Each of these methods of agitation were performed in combination with three rinse (extraction) fluids: phosphate buffer (Butterfield’s buffer), phosphate buffer with 0.2% polysorbate 80 (a surfactant), and water with 1% peptone and 1% polysorbate 80 (Fluid D). The highest recovery efficiencies were observed with sonication plus mechanical shaking; of the three extraction media, Fluid D gave the highest first-rinse recovery efficiency (65%) and Butterfield’s buffer gave the lowest (39%). Each of the three recovery methods, however appeared to reach “exhaustion”, a subsequent rinse giving less than 10% of the recovery found in the first rinse. The results demonstrated the importance of performing bioburden method development and validation studies. The method validation strategy described here, using a combination of tissue inoculation and repetitive extraction, showed the superiority of sonication plus mechanical shaking using Fluid D as the rinse medium. In addition, the use of only the exhaustive extraction approach could have resulted in the development of a methodology that consistently underestimated the bioburden present on/in recovered tissue.     

Processing of immunoisolated pancreatic islets: implications for histological analyses of hydrated tissue

Routine tissue processing is usually associated with histological artifacts as a consequence of shrinkage and distortion during dehydration required for embedding. With hydrated specimens such as lung, embryonic, and tissues in hydrophilic membranes, tissue processing can induce severe artifacts that interfere with adequate microscopic evaluation. Here we present a method for embedding hydrophilic alginate-polylysine microencapsulated pancreatic tissue that combines the absence of histological artifacts with a practical tissue processing method. We found that the glycol-methacrylate (GMA)-embedding method preserved the integrity of the encapsulated tissue better than snap-freezing or paraffin embedding, but the overall quality of the hydrophilic capsules remained poor Next, we modified the GMA method by introducing gradual dehydration to investigate whether the integrity of the sectioned capsules was better maintained by a more gradual pattern of water extraction. This modification resulted in well-preserved morphological details of the hydrophilic membranes, hydrogel-cell interface, and encapsulated pancreatic tissue. Subsequent routine staining gave excellent contrast between the islet tissue and hydrophilic components, which allowed adequate quantitative histological and pathological comparisons.     

A stochastic bioburden model for spacecraft sterilization

An important factor in the calculation of sterilization cycles for spacecraft is the bioburden on the surface of the spacecraft at the start of the cycle. This bioburden must be predicted by the use of models. This paper presents a stochastic model for the prediction of the bioburden on the surfaces of spacecraft. This model has many desirable properties as well as being consistent with observations.This work was conducted under Contract No. W-12,853, Planetary Programs, Office, of Space Science and Applications, NASA Headquarters, Washington, D.C.     

Extreme Spore UV Resistance of <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Isolates Obtained from an Ultraclean Spacecraft Assembly Facility

Recent environmental microbial sampling of the ultraclean Spacecraft Assembly Facility at NASA Jet Propulsion Laboratory (JPL-SAF) identified spores of Bacillus pumilus as major culturable bacterial contaminants found on and around spacecraft. As part of an effort to assess the efficacy of various spacecraft sterilants, purified spores of 10 JPL-SAF B. pumilus isolates were subjected to 254-nm UV and their UV resistance was compared to spores of standard B. subtilis biodosimetry strains. Spores of six of the 10 JPL-SAF isolates were significantly more resistant to UV than the B. subtilis biodosimetry strain, and one of the JPL-SAF isolates, B. pumilus SAFR-032, exhibited the highest degree of spore UV resistance observed by any Bacillus spp. encountered to date.     

Detection of Frequency Resonance Energy Transfer Pair on Double-Labeled Microsphere and Bacillus anthracis Spores by Flow Cytometry

Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare. Fluorescence methods combined with immunodiagnostic methods are the most common. To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method. FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET. Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method. This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system. As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores. The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B. anthracis exosoporium components. The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor. On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (E^q = 35%) and appearance of the alexa594 fluorescence (E^s = 22%), as detected by flow cytometry analysis. The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B. thuringiensis subsp. israelensis and B. subtilis. This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B. anthracis spores by a factor of 10 with respect to B. thuringiensis subsp. israelensis and a factor of 100 with respect to B. subtilis as control spores.     

Development of a simple embedding procedure allowing immunocytochemical localization at the ultrastructural level

Immunobed solution A is a water-soluble acrylic compound recently developed for immunocytochemical localization at the light microscopic level. In this study, we combined it with methyl methacrylate (MMA) to achieve sufficient hardness to obtain ultra-thin sections. Samples of platelets were dehydrated and embedded in the water-soluble acrylic mixture (WSAM). The embedding process was carried out at 4 degrees C and final polymerization was induced with either chemical (benzoyl peroxide) or physical (UV light) catalysts. Tubulin was localized at the ultrastructural level in sections embedded according to these two methods. Results were compared with those obtained in platelets processed in Lowicryl. Dehydration and embedding with the WSAM yielded a preservation of antigenicity similar to that obtained in Lowicryl. The new procedure benefits from the low temperature achieved during polymerization, providing good ultrastructural morphology and immunolocalization of protein antigens with the simplicity of a routine embedding procedure for light microscopy.     

Evaluation of alcohol wipes used during aseptic manufacturing

Aim: During aseptic manufacturing and specifically during the transfer of items into an isolator, disinfection of surfaces is essential for reducing the risk of final product contamination. Surface disinfection can be carried out by a variety of methods, however the most accepted current practice is a combination of spraying with 70% alcohol and wiping. The aim of this study was to evaluate the effectiveness of two wipe systems by determining their ability to remove, kill and transfer bacterial contaminants from standardized surfaces. Methods and Results: The protocol used to achieve these objectives was based on a newly published method specifically designed to test wipes. Alcohol impregnated wipes performed better at reducing microbial bioburden than the alcohol spray/dry wipe applications. Impregnated wipes drastically reduced (1–2 log₁₀ reduction) a small bioburden (approx. 2 log₁₀) of spores of Bacillus subtilis and methicillin‐resistant Staphylococcus aureus from the surface, but failed to remove (<0·2 log₁₀ reduction) Staphylococcus epidermidis. The alcohol spray/dry wipes did not manage to remove (<0·2 log₁₀ reduction) spore or bacterial bioburden from surfaces and was able to transfer some viable micro‐organisms to other surfaces. Both wipe types showed poor antimicrobial efficacy (<1 log₁₀ reduction) against the test bacteria and spores. Conclusions: As far as the authors are aware this is the first time that such a practical study has been reported and our results suggest that the best wipes for surface disinfection in aseptic units are the alcohol (IPA) impregnated wipes when compared with the dry wipes sprayed with alcohol. Significance and Impact of the Study: The impregnated wipes performed better than the dry wipes sprayed with alcohol and should be used for surface disinfection in aseptic units.     

Release of Small Polyuronides from Nitella Cell Walls during Ionic Exchange

Mono-divalent ion exchange in isolated cell walls of Nitella flexilis (L.) Ag. induces a marked loss of wall polymers and a decrease in the wall cationic exchange capacity. These data correlate with the replacement in the walls of adsorbed Mn²⁺ by Na⁺ ions. Boiling wall samples in methanol for 1 h or keeping the ionic solutions chilled to 4°C does not inhibit the cell wall polymer leakage but modifies the kinetics both of the ionic exchange and of the released polymers. These data are more compatible with physical rather than enzymic induced processes. The extracted polymers in the successively renewed NaCl solutions initially belong to the wall protein and pectin fractions and mainly to pectic fractions subsequently. Determination of the average degree of polymerization shows that the average molecular size of the lost acidic polysaccharides increases with extraction time up an average polymerization degree of 25. Enzyme-linked immunosorbent assay inhibition tests show the presence of homopolymer blocks equal to or higher than 10 in the released polymer fragments. Compositional analysis of released polysaccharides suggests that the pectin lost by action of monovalent ions was largely composed of rhamnogalacturonans whose acidic residue fraction is approximately 60% in association with galactose chains. Small quantities of glucuronylated xylans are also found.     

Immunodetection of low concentrations of ovalbumin in cryoprocessed quail oviduct cells by semi-automatic quantitative analysis

Summary— In a previous paper (Quintana et al, Biol Cell 72, (1991) 167–180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significantly higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.     

Influence of polymerization temperature on the molecular recognition of imprinted polymers

This paper aimed at investigating the influence of polymerization temperature on the molecular recognition of molecularly imprinted polymers (MIPs) based on multiple non-covalent interactions. 3-l-Phenylalanylaminopyridine (3-l-PheNHPy) imprinted polymers were prepared using azobisnitriles as either thermal initiators or photoinitiators at various temperatures of 10, 40 and 60 degrees C, respectively. These polymers were subsequently evaluated in the high-performance liquid chromatographic (HPLC) mode for enantioselectivity. An unexpected result shows that polymer prepared at 40 degrees C has the highest enantioselectivity, but not the polymer prepared at lower temperature of 10 degrees C. Further, the effect of elution temperature and sample load on the selectivity of polymers was investigated in detail. In order to get a better understanding of the "exception", the influence of polymerization temperature on the polymerization extent and polymer morphology was studied by FT-IR spectrum test, cross-polarization magic angle spinning (CP-MAS) (13)NMR spectra experiment and pore analysis. Based on these results we attribute this "exception" to that there is a tradeoff between the extent of polymerization and stabilization of the template-functional monomer complexes. And an optimal polymerization temperature can be found for each combination of template and monomer.     

Universal metastability of sickle hemoglobin polymerization

Sickle hemoglobin (HbS) polymerization occurs when the concentration of deoxyHbS exceeds a well-defined solubility. In experiments using sickle hemoglobin droplets suspended in oil, it has been shown that when polymerization ceases the monomer concentration is above equilibrium solubility. We find that the final concentration in uniform bulk solutions (i.e., with negligible boundaries) agrees with the droplet measurements, and both exceed the expected solubility. To measure hemoglobin in uniform solutions, we used modulated excitation of trace amounts of CO in gels of HbS. In this method, a small amount of CO is introduced to a spatially uniform deoxyHb sample, so that less than 2% of the sample is liganded. The liganded fraction is photolyzed repeatedly and the rate of recombination allows the concentration of deoxyHbS in the solution phase to be determined, even if polymers have formed. Both uniform and droplet samples exhibit the same quantitative behavior, exceeding solubility by an amount that depends on the initial concentration of the sample, as well as conditions under which the gel was formed. We hypothesize that the early termination of polymerization is due to the obstruction in polymer growth, which is consistent with the observation that pressing on slides lowers the final monomer concentration, making it closer to solubility. The thermodynamic solubility in free solution is thus achieved only in conditions with low polymer density or under external forces (such as found in sedimentation) that disrupt polymers. Since we find that only about 67% of the expected polymer mass forms, this result will impact any analysis predicated on predicting the polymer fraction in a given experiment.     

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, α,ω-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of α-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The α,ω-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-∈-amino caproic acid, by DCC coupling, resulting in the formation of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, ¹H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.     

Molecular imprinting and solid phase extraction of flavonoid compounds

Molecularly imprinted polymers (MIPs) for quercetin have been successfully prepared by a thermal polymerization method using 4-vinylpyridine (4-VP) and ethylene glycol dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The obtained molecularly imprinted polymers were evaluated by HPLC using organic eluents, with respect to their selective recognition properties for quercetin and related compounds of the flavonoid class. Two equivalent control polymers, a blank polymer and a polymer imprinted with a structural analogous template, were synthesized, in order to confirm the obtained results. Furthermore, preliminary experiments confirm the applicability of the prepared MIPs for solid phase extraction (SPE), as rapid and facile clean-up of wine samples for HPLC analysis is an envisaged field of application. The successful preparation of molecularly imprinted polymers for flavones provides an innovative opportunity for the development of advanced separation materials, with applications in the field of wine and fermentation analysis.     

Horseradish peroxidase embedded in polyacrylamide nanoparticles enables optical detection of reactive oxygen species

We have synthesized and characterized new nanometer-sized polyacrylamide particles containing horseradish peroxidase and fluorescent dyes. Proteins and dyes are encapsulated by radical polymerization in inverse microemulsion. The activity of the encapsulated enzyme has been examined and it maintains its ability to catalyze the oxidation of guaiacol with hydrogen peroxide as the electron acceptor, although at a slightly lower rate compared to that of the free enzyme in solution. The embedded enzyme is also capable of catalyzing the peroxidase-oxidase reaction. However, the rate is decreased by a factor of 2-3 compared to that of the free enzyme. The reduced rate is probably due to limitation of diffusion of substrates and products into and out of the particles. The catalytic activity of horseradish peroxidase in the polyacrylamide matrix demonstrates that the particles have pores which are large enough for substrates to enter and products to leave the polymer matrix containing the enzyme. The polymer matrix protects the embedded enzyme from proteolytic digestion, which is demonstrated by treating the particles with a mixture of the two proteases trypsin and proteinase K. The particles allow for quantification of hydrogen peroxide and other reactive oxygen species in microenvironments, and we propose that the particles may find use as nanosensors for use in, e.g., living cells.     

Direct detection of analyte binding to single molecularly imprinted polymer particles by confocal Raman spectroscopy

We describe the use of Raman spectroscopy to detect and quantify, for the first time, the presence of the imprinting template in single molecularly imprinted polymer microspheres. The polymers were imprinted with the β-blocking drugs propranolol and atenolol, and precipitation polymerization was used to obtain spherical particles of diameters of 200 nm and 1.5 μm. The size of the Raman laser spot being between 1 μm and a few μm, the nanoparticles were used for bulk detection whereas with micrometer-sized particles, quantitative measurements on single particles were possible. The laser power, and consequently the acquisition times, needed to be adapted as a function of the polymer and template used in order to avoid burning. Analyte quantification from Raman spectra is straightforward by determining the peak height of a typical Raman band of the analyte, and by using a typical polymer peak for normalization. Relatively low detection limits down to 1 μM have been reached for the detection of S-propranolol through bulk measurements on MIP nanoparticles.