Colorimetricdetection of penicillin was performed with enzme-based biosensors employingTMV-derived nanoadapter scaffolds. Streptavidin conjugation of a commercialpenicillinase and its binding to biotin-linked TMV rods enabled selectivehigh-surface density immobilization. Thecolorimetric detection of penicillinase activity is based on acidometry employingthe hydrolysis of penicillin with differential halochromic color changes of pHindicators (Figure 1C). To identify the most suitable pH indicator, six pHindicator dyes were tested: phenol red, bromcresol purple, methyl red,bromthymol blue, cresol red and phenolphthalein, which showed a clear colorchange between pH 3 and pH 10 (the expected working range of thepenicillinase biosensor) (Figure S1). To quantify the proton generation, the absorptionspectra of each pH indicator between pH 1 and pH 12 were measured todetermine the wavelength of maximal change (Table 2, Figure S2). Phenolphthaleindid not show any relevant color change in the analyte/enzyme mixtures. Best changes(OD/min) (Figure S3) and ?OD values (ODstart – ODend) (Table2) were obtained by bromcresol purple (?OD = 0.
76), followed by phenolred (?OD = 0.36), cresol red, methyl red and bromthymol blue (?OD valuesfrom 0.1 to 0.
17). With bromcresol purple, low enzyme amounts (5 ng) in lowpenicillin concentrations (5 mM penG) were detectable (Figure S3). The enzymepenicillinase was further tested for its ability to detect different ?-lactamantibiotics. A spectrum of six different antibiotics was tested: penicillin G(benzylpenicillin), tricarcillin (carboxypenicillin), carbenicillin(carboxypenicillin), ampicillin (aminopenicillin), cloxacillin and cefotexim(cephalosporine) (Figure S5). Penicillin G and tricarcillin (semisynthetic) areinjectable antibiotics in human medizine, carbenicillin and ampicillin are semisyntheticbroad-spectrum antibiotic, cloxacillin is a ?-lactamase resistant antibioticsolely used in veterinary medicine, cefotaxim belongs to third generation broad-spectrumcephalosporins.
As expected, penicillinase was not able to detect cloxacillinand cefotaxime, due to the inhibited hydrolysis of the ?-lactam ring. However,low absorbance changes in approaches with 100 mU enzyme allowed detecten ofmoderate ampicillin concentrations (Figure S5, B). Carbenicillin andtricarcillin were easily detectable, however lower absorbance changes wereobtained due to the semisynthetic modifications of the ?-lactam backbone. Thepenicillinase, used in this study, is able to detect at least 3 different?-lactam antibiotics; however, it is very likely that Pen is also able todetect a much higher number of natural and semisynthetic penicillin-derivates. Adsorptivebinding of biotinylated TMV to the sensor surfaces of microtiter plate wells wasfollowed by enzyme immobilization by streptavidin-biotin binding (Figure 2A). Tothis aim, the most surface-accessible cysteine residues of the TMVCys particleswere modified with maleimide-PEG11-biotin linker molecules (TMVCys/Bio),with a biotinylation efficiency of 95 % corresponding to ?2000 biotinanchors per virus rod (Figure S4A). To allow a strong and specific couplingof Pen, enzyme molecules were conjugated with streptavidin (SA)-complexes,resulting in SA-Pen (conjugation effiency: > 90 % of the Penmolecules (Figure S4B)).
Thepenicillinase activity was not impaired by the streptavidin conjugation, since 60 µUof both SA-conjugated and free penicillinase in solution yielded equal absorptionchanges of ?3 OD/min in the reaction mixture (Figure 2A). On polystyrenemicrotiter plates immobilized TMVCys/Bio sticks demonstrated the requirementof streptavidin for successful coupling of penicillinase. Both biotin equipmentof the TMV adapters as well as streptavidin conjugation of the penicillinasewas crucial for immobilization of significant amounts of enzymes, resulting in detectablesubstrate conversion rates (Figure 2A). The successful enzyme decorationof TMVCys/Bio sticks was verified by TEM of freely suspendedparticles prepared in parallel. Enzyme conjugation to TMV adapter sticks was evidencedby an increased diameter of 30 – 33 nm and intensified staining forthe TMV adapter sticks (Figure 2B). Thecatalytic activities of Pen and SA-Pen (measured as OD/min) were analyzed afterimmobilization on solid supports in direct comparison between setups based ondirect (adsorptive, without carrier) binding and setups equipped withbiotinylated TMV nano-carriers as adapters. Equal input amounts of SA-Pen orPen (each 1 U) were applied for binding into microtiter plate cavities asdepicted in Figure 3: TMVCys/Bio tubes (No.
1, 5)(5 µg), TMVCys/Bio tubes (5 µg) followed by BSA treatmentto reduce unspecific enzyme binding (No. 2, 6), bare plate cavities (No. 3, 7)or cavities pretreated with BSA (No. 4, 8; see also Figure 3C). Followingoptional TMV adapter binding, optional BSA blocking, subsequent incubation withenzyme or enzyme SA-conjugate, and removal of unbound SA-Pen or Penthereafter, absorption changes reflecting the enzyme activity in each approach weredetermined by spectrophotometry.
Highest absorption changes over time wereobtained in TMV-assisted layouts enabling SA-Pen bioaffinity coupling.Cavities equipped with TMVCys/Bio (No. 1) reached a maximum changeof 13 × 10-2 OD/min (Figure 3A),due to enzyme immobilization primarily through biotin-streptavidin interaction.The use of TMVCys/Bio and BSA blocking (No. 2) achieved aslightly lower absorption change of 11 x 10?2 OD/min,indicating that in this layout, solely biotin-specific immobilization of SA?Penoccurred (and thus, a minor additional amount of enzyme had adsorbed to thenon-blocked polystyrene surface in layout No. 1).
The blocking efficiencyof BSA was confirmed in the cavities with BSA blocking preceding enzymeincubation (No. 4, 8), which did not exhibit any enzyme activityafter washing. In layouts lacking either the viral adapter (No. 3, 7),or streptavidin-conjugation of the enzyme (No. 5, 6), only lowerabsorption changes of around 8-9 x 10-2 OD/min wereobtained, underlining the efficiency of the TMV-biotin-streptavidin enzyme trappingmethod.