Ultrafast terahertz Stark spectroscopy reveals the excited-state dipole moments of retinal in bacteriorhodopsin.

The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR). Extensive experimental and theoretical work has addressed excited-state dynamics and isomerization via a conical intersection with the ground state. In conflicting molecular pictures, the excited state potential energy surface has been modeled as a pure S[Formula: see text] state that intersects with the ground state, or in a 3-state picture involving the S[Formula: see text] and S[Formula: see text] states. Here, the photoexcited system passes two crossing regions to return to the ground state. The electric dipole moment of the Schiff base in the S[Formula: see text] and S[Formula: see text] state differs strongly and, thus, its measurement allows for assessing the character of the excited-state potential. We apply the method of ultrafast terahertz (THz) Stark spectroscopy to measure electric dipole changes of wild-type BR and a BR D85T mutant upon electronic excitation. A fully reversible transient broadening and spectral shift of electronic absorption is induced by a picosecond THz field of several megavolts/cm and mapped by a 120-fs optical probe pulse. For both BR variants, we derive a moderate electric dipole change of 5 [Formula: see text] 1 Debye, which is markedly smaller than predicted for a neat S[Formula: see text]-character of the excited state. In contrast, S[Formula: see text]-admixture and temporal averaging of excited-state dynamics over the probe pulse duration gives a dipole change in line with experiment. Our results support a picture of electronic and nuclear dynamics governed by the interaction of S[Formula: see text] and S[Formula: see text] states in a 3-state model.

Nanostructured lipopeptide-based membranomimetics for stabilizing bacteriorhodopsin.

Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or ß-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.

Membrane Proteins in Action Monitored by pH-Responsive Liquid Crystal Biosensors.

Liquid crystal (LC) biosensors have received significant attention for their potential applications for point-of-care devices due to their sensitivity, low cost, and easy read-out. They have been employed to detect a wide range of important biological molecules. However, detecting the function of membrane proteins has been extremely challenging due to the difficulty of integrating membrane proteins, lipid membranes, and LCs into one system. In this study, we addressed this challenge by monitoring the proton-pumping function of bacteriorhodopsin (bR) using a pH-sensitive LC thin film biosensor. To achieve this, we deposited purple membranes (PMs) containing a 2D crystal form of bRs onto an LC-aqueous interface. Under light, the PM patches changed the local pH at the LC-aqueous interface, causing a color change in the LC thin film that is observable through a polarizing microscope with crossed polarizers. These findings open up new opportunities to study the biofunctions of membrane proteins and their induced local environmental changes in a solution using LC biosensors.

Rigid Cyclic Fluorinated Detergents: Fine-Tuning the Hydrophilic-Lipophilic Balance Controls Self-Assembling and Biochemical Properties.

We report herein the synthesis of three detergents bearing a perfluorinated cyclohexyl group connected through a short, hydrogenated spacer (i.e., propyl, butyl, or pentyl) to a ß-maltoside polar head that are, respectively, called FCymal-3, FCymal-4, and FCymal-5. Increasing the length of the spacer decreased the critical micellar concentration (CMC), as demonstrated by surface tension (SFT) and isothermal titration calorimetry (ITC), from 5 mM for FCymal-3 to 0.7 mM for FCymal-5. The morphology of the micelles was studied by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS), indicating heterogeneous rod-like shapes. While micelles of FCymal-3 and -4 have similar hydrodynamic diameters of ∼10 nm, those of FCymal-5 were twice as large. We also investigated the ability of the detergents to solubilize lipid membranes made of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC). Molecular modeling indicated that the FCymal detergents generate disorder in lipid bilayers, with FCymal-3 being inserted more deeply into bilayers than FCymal-4 and -5. This was experimentally confirmed using POPC vesicles that were completely solubilized within 2 h with FCymal-3, whereas FCymal-5 required >8 h. A similar trend was noticed for the direct extraction of membrane proteins from E. coli membranes, with FCymal-3 being more potent than FCymal-5. An opposite trend was observed in terms of stabilization of the two model membrane proteins bacteriorhodopsin (bR) and SpNOX. In all three FCymal detergents, bR was stable for at least 2 months with no signs of aggregation. However, while the structural integrity of bR was fully preserved in FCymal-4 and -5, minor bleaching was observed in FCymal-3. Similarly, SpNOX exhibited the least activity in FCymal-3 and the highest activity in FCymal-5. By combining solubilizing and stabilizing potency, FCymal detergents push forward our expectations of the usefulness of fluorinated detergents for handling and investigating membrane proteins.

Uniaxial Symmetry Breaking in Bacteriorhodopsin at the Thermal Phase Transition of Lipids of Purple Membranes.

The article correlates between symmetry breaking and phase transition. An analogy, extending from physics to biology, is known to exist between these two topics. Bacteriorhodopsin (bR) as a paradigm of membrane proteins has been used as a case study in the present work. The bR, as the sole protein embedded in what is called a purple membrane (PM), has attracted widespread interest in bionanotechnological applications. The lipids of PM have a crucial role in maintaining the crystal lattice of bR inside PM. For this reason, the present work has been concerned with elucidating the thermal phase transition properties of the PM lipids in orthogonal directions. The results indicated that the axial symmetry of bR exhibits considerable changes occurring at the thermal phase transition of lipids. These changes are brought by an anomaly observed in the time course of orthogonal electric responses during the application of thermal fields on PM. The observed anomaly may bear on symmetry breaking in bR occurring at the phase transition of lipids based on such analogy found between symmetry breaking and phase transition. Lipid-protein interactions may underlie the broken axial symmetry of bR at such lipid thermal transition of PM. Accordingly, thermally perturbed axial symmetry of bR may be of biological relevance relying on the essence of the crystal lattice of bR. Most importantly, a question has to be raised in the present study: Can bR, as a helical protein with broken axial symmetry, affect the symmetry breaking of helical light? This may be of potential technical applications based on a recent discovery that bR breaks the symmetry of helical light.

Helical reorganization in the context of membrane protein folding: Insights from simulations with bacteriorhodopsin (BR) fragments.

Membrane protein folding is distinct from folding of soluble proteins. Conformational acquisition in major membrane protein subclasses can be delineated into insertion and folding processes. An exception to the "two stage" folding, later developed to "three stage" folding, is observed within the last two helices in bacteriorhodopsin (BR), a system that serves as a model membrane protein. We employ a reductionist approach to understand interplay of molecular factors underlying the apparent defiance. Leveraging available solution NMR structures, we construct, sample in silico, and analyze partially (PIn) and fully inserted (FIn) BR membrane states. The membrane lateral C-terminal helix (CH) in PIn is markedly prone to transient structural distortions over microsecond timescales; a disorder prone region (DPR) is thereby identified. While clear transmembrane propensities are not acquired, the distortions induce alterations in local membrane curvature and area per lipid. Importantly, energetic decompositions reveal that overall, the N-terminal helix (NH) is thermodynamically more stable in the PIn. Higher overall stability of the FIn arises from favorable interactions between the NH and the CH. Our results establish lack of spontaneous transition of the PIn to the FIn, and attributes their partitioning to barriers that exceed those accessible with thermal fluctuations. This work paves the way for further detailed studies aimed at determining the thermo-kinetic roles of the initial five helices, or complementary external factors, in complete helical folding and insertion in BR. We comment that complementing such efforts with the growing field of machine learning assisted energy landscape searches may offer unprecedented insights.

Effects of stand-alone polar residue on membrane protein stability and structure.

Helical membrane proteins generally have a hydrophobic nature, with apolar side chains comprising the majority of the transmembrane (TM) helices. However, whenever polar side chains are present in the TM domain, they often exert a crucial role in structural interactions with other polar residues, such as TM helix associations and oligomerization. Moreover, polar residues in the TM region also often participate in protein functions, such as the Schiff base bonding between Lys residues and retinal in rhodopsin-like membrane proteins. Although many studies have focused on these functional polar residues, our understanding of stand-alone polar residues that are energetically unfavored in TM helixes is limited. Here, we adopted bacteriorhodopsin (bR) as a model system and systematically mutated 17 of its apolar Leu or Phe residues to polar Asn. Stability measurements of the resulting mutants revealed that all of these polar substitutions reduced bR stability to various extents, and the extent of destabilization of each mutant bR is also correlated to different structural factors, such as the relative accessible surface area and membrane depth of the mutation site. Structural analyses of these Asn residues revealed that they form sidechain-to-backbone hydrogen bonds that alleviate the unfavorable energetics in hydrophobic and apolar surroundings. Our results indicate that membrane proteins are able to accommodate certain stand-alone polar residues in the TM region without disrupting overall structures.

Retinal photoisomerization versus counterion protonation in light and dark-adapted bacteriorhodopsin and its primary photoproduct.

Discovered over 50 years ago, bacteriorhodopsin is the first recognized and most widely studied microbial retinal protein. Serving as a light-activated proton pump, it represents the archetypal ion-pumping system. Here we compare the photochemical dynamics of bacteriorhodopsin light and dark-adapted forms with that of the first metastable photocycle intermediate known as "K". We observe that following thermal double isomerization of retinal in the dark from bio-active all-trans 15-anti to 13-cis, 15-syn, photochemistry proceeds even faster than the ~0.5 ps decay of the former, exhibiting ballistic wave packet curve crossing to the ground state. In contrast, photoexcitation of K containing a 13-cis, 15-anti chromophore leads to markedly multi-exponential excited state decay including much slower stages. QM/MM calculations, aimed to interpret these results, highlight the crucial role of protonation, showing that the classic quadrupole counterion model poorly reproduces spectral data and dynamics. Single protonation of ASP212 rectifies discrepancies and predicts triple ground state structural heterogeneity aligning with experimental observations. These findings prompt a reevaluation of counter ion protonation in bacteriorhodopsin and contribute to the broader understanding of its photochemical dynamics.

Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy.

Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification. Here, we measured these energetics on the millisecond time scale using atomic-force-microscopy-based single-molecule force spectroscopy. Precisely, timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8-amino-acid region of bR's G-helix. These dynamics changed when the EF-helix pair moved ~9 Å away from this end of the G helix during the "open" portion of bR's photocycle. In ~60% of the data, we observed abrupt light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pH-dependence of this destabilization were consistent with prior measurements of bR's open phase. The frequency of light-induced destabilization increased with the duration of illumination and was dramatically reduced in the triple mutant (D96G/F171C/F219L) thought to trap bR in its open phase. In the other ~40% of the data, photoexcitation unexpectedly stabilized a longer-lived putative misfolded state. Through this work, we establish a general single-molecule force spectroscopy approach for measuring ligand-induced energetics and lifetimes in membrane proteins.

Protein-lipid acyl chain interactions: Depth-dependent changes of segmental mobility of phospholipid in contact with bacteriorhodopsin.

Boundary lipids surrounding membrane proteins play an essential role in protein function and structure. These protein-lipid interactions are mainly divided into electrostatic interactions between the polar amino acids of proteins and polar heads of phospholipids, and hydrophobic interactions between protein transmembrane sites and phospholipid acyl chains. Our previous report (Kawatake et al., Biochim. Biophys. Acta 1858 [2016] 2106-2115) covered a method for selectively analyzing boundary lipid interactions and showed differences in membrane protein-peripheral lipid interactions due to differences in their head group. Interactions in the hydrophobic acyl chains of phospholipids are relatively consistent among proteins, but the details of these interactions have not been elucidated. In this study, we reconstituted bacteriorhodopsin as a model protein into phospholipid membranes labeled with 2H and 13C for solid-state NMR measurement to investigate the depth-dependent effect of the head group structure on the lipid bilayer. The results showed that the position of the phospholipid near the carbonyl carbon was affected by the head group in terms of selectivity for protein surfaces, whereas in the deep interior of the bilayer near the leaflet interface, there was little difference between the head groups, indicating that the dependence of their interactions on the head group was much reduced.

Light Color-Controlled pH-Adjustment of Aqueous Solutions Using Engineered Proteoliposomes.

Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light-driven proton pumps bacteriorhodopsin and blue light-absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer-controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH-sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color-controlled pH-adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications.

Cis-Trans Reisomerization Preceding Reprotonation of the Retinal Chromophore Is Common to the Schizorhodopsin Family: A Simple and Rational Mechanism for Inward Proton Pumping.

The creation of unidirectional ion transporters across membranes represents one of the greatest challenges in chemistry. Proton-pumping rhodopsins are composed of seven transmembrane helices with a retinal chromophore bound to a lysine side chain via a Schiff base linkage and provide valuable insights for designing such transporters. What makes these transporters particularly intriguing is the discovery of both outward and inward proton-pumping rhodopsins. Surprisingly, despite sharing identical overall structures and membrane topologies, these proteins facilitate proton transport in opposite directions, implying an underlying rational mechanism that can transport protons in different directions within similar protein structures. In this study, we unraveled this mechanism by examining the chromophore structures of deprotonated intermediates in schizorhodopsins, a recently discovered subfamily of inward proton-pumping rhodopsins, using time-resolved resonance Raman spectroscopy. The photocycle of schizorhodopsins revealed the cis-trans thermal isomerization that precedes reprotonation at the Schiff base of the retinal chromophore. Notably, this order has not been observed in other proton-pumping rhodopsins, but here, it was observed in all seven schizorhodopsins studied across the archaeal domain, strongly suggesting that cis-trans thermal isomerization preceding reprotonation is a universal feature of the schizorhodopsin family. Based on these findings, we propose a structural basis for the remarkable order of events crucial for facilitating inward proton transport. The mechanism underlying inward proton transport by schizorhodopsins is straightforward and rational. The insights obtained from this study hold great promise for the design of transmembrane unidirectional ion transporters.

A light-sensitive protein-based wearable pH biometer.

Bacteriorhodopsin is a biological material with excellent photosensitivity properties. It can directly convert optical signals into electrical signals and is widely used in various biosensors. Here, we present a bR-based wearable pH biometer that can be used to monitor wound infection. The mechanism of the pH-sensitive effect of the bR electrode is explained, which generates a transient photovoltage under light irradiation and a negative photovoltage when the lamp is turned off. Since the photoelectric signal of bR is affected by different pH values, the photovoltage is changed by adjusting the pH value. The ratio (Vn/Vp) of negative photovoltage (Vn) to positive photovoltage (Vp) has a good linear relationship (R2 = 0.9911) in the pH range of 4.0-10.0. In vitro experiments using rats as a model confirmed that this wearable pH biometer can monitor pH changes that occur in wound infection.

Photosynthetic Protein-Based Retinal Ganglion Cell Receptive Fields for Detecting Edges and Brightness Illusions.

Bacteriorhodopsin, isolated from a halophilic bacterium, is a photosynthetic protein with a structure and function similar to those of the visual pigment rhodopsin. A voltaic cell with bacteriorhodopsin sandwiched between two transparent electrodes exhibits a time-differential response akin to that observed in retinal ganglion cells. It is intriguing as a means to emulate excitation and inhibition in the neural response. Here, we present a neuromorphic device emulating the retinal ganglion cell receptive field fabricated by patterning bacteriorhodopsin onto two transparent electrodes and encapsulating them with an electrolyte solution. This protein-based artificial ganglion cell receptive field is characterized as a bandpass filter that simultaneously replicates excitatory and inhibitory responses within a single element, successfully detecting image edges and phenomena of brightness illusions. The device naturally emulates the highly interacting ganglion cell receptive fields by exploiting the inherent properties of proteins without the need for electronic components, bias power supply, or an external operating circuit.

Features of the Mechanism of Proton Transport in ESR, Retinal Protein from Exiguobacterium sibiricum.

Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.

Similarities and Differences in Photochemistry of Type I and Type II Rhodopsins.

The diversity of the retinal-containing proteins (rhodopsins) in nature is extremely large. Fundamental similarity of the structure and photochemical properties unites them into one family. However, there is still a debate about the origin of retinal-containing proteins: divergent or convergent evolution? In this review, based on the results of our own and literature data, a comparative analysis of the similarities and differences in the photoconversion of the rhodopsin of types I and II is carried out. The results of experimental studies of the forward and reverse photoreactions of the bacteriorhodopsin (type I) and visual rhodopsin (type II) rhodopsins in the femto- and picosecond time scale, photo-reversible reaction of the octopus rhodopsin (type II), photovoltaic reactions, as well as quantum chemical calculations of the forward photoreactions of bacteriorhodopsin and visual rhodopsin are presented. The issue of probable convergent evolution of type I and type II rhodopsins is discussed.

Detection of Lipid-Bound Bacteriorhodopsin Trimer Complex Directly from Purple Membrane by Native Mass Spectrometry.

Native mass spectrometry (MS) was used to detect the membrane protein, bacteriorhodopsin (bR), in its 27 kDa monomeric form and trimeric assemblies directly from lipid-containing purple membranes (PMs) from the halophilic archaeon, Halobacterium salinarum. Trimer bR ion populations bound to lipid molecules were detected with n-octyl ß-d-glucopyranoside as the solubilizing detergent; the use of octyl tetraethylene glycol monooctyl ether or n-dodecyl-ß-d-maltopyranoside resulted in only detection of monomeric bR. The archaeal lipids phosphotidylglycerolphosphate methyl ester and 3-HSO3-Galp-ß1,6-Manp-α1,2-Glcp-α1,1-sn-2,3-diphytanylglycerol were the only lipids in the PMs found to bind to bR, consistent with previous high-resolution structural studies. Removal of the lipids from the sample resulted in the detection of only the bR monomer, highlighting the importance of specific lipids for stabilizing the bR trimer. To the best of our knowledge, this is the first report of the detection of the bR trimer with resolved lipid-bound species by MS.

Peptide-Coated Bacteriorhodopsin-Based Photoelectric Biosensor for Detecting Rheumatoid Arthritis.

An effective early diagnosis is important for rheumatoid arthritis (RA) management. This study reveals a novel RA detection method using bacteriorhodopsin as a photoelectric transducer, a light-driven proton pump in purple membranes (PMs). It was devised by covalently conjugating a PM monolayer-coated electrode with a citrullinated-inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3)542-556 peptide that recognizes the serum RA-associated autoantibodies. The direct serum coating decreased the photocurrents in the biosensor, with the reduction in the photocurrent caused by coating with an RA-patient serum that is significantly larger than that with a healthy-control serum (38.1% vs. 20.2%). The difference in the reduction in the photocurrent between those two serum groups widened after the serum-coated biosensor was further labeled with gold nanoparticle (AuNP)-conjugated anti-IgA (anti-IgA-AuNP) (53.6% vs. 30.6%). Both atomic force microscopic (AFM) and Raman analyses confirmed the sequential peptide, serum, and anti-IgA-AuNP coatings on the PM-coated substrates. The reductions in the photocurrent measured in both the serum and anti-IgA-AuNPs coating steps correlated well with the results using commercial enzyme-linked immunosorbent assay kits (Spearman rho = 0.805 and 0.787, respectively), with both a sensitivity and specificity close to 100% in both steps. It was shown that an RA diagnosis can be performed in either a single- or two-step mode using the developed biosensor.

Quantifying Bacteriorhodopsin Activity as a Function of its Local Environment with a Raman-Based Assay.

Bacteriorhodopsin (bR) is a transmembrane protein that functions as a light-driven proton pump in halophilic archaea. The bR photocycle has been well-characterized; however, these measurements almost exclusively measured purified bR, outside of its native membrane. To investigate what effect the cellular environment has on the bR photocycle, we have developed a Raman-based assay that can monitor the activity of the bR in a variety of conditions, including in its native membrane. The assay uses two continuous-wave lasers, one to initiate photochemistry and one to monitor bR activity. The excitation leads to the steady-state depletion of ground-state bR, which directly relates to the population of photocycle intermediate states. We have used this assay to monitor bR activity both in vitro and in vivo. Our in vitro measurements confirm that our assay is sensitive to bulk environmental changes reported in the literature. Our in vivo measurements show a decrease in bR activity with increasing extracellular pH for bR in its native membrane. The difference in activity with increasing pH indicates that the native membrane environment affects the function of bR. This assay opens the door to future measurements into understanding how the local environment of this transmembrane protein affects function.

Small-Molecule Analysis Based on DNA Strand Displacement Using a Bacteriorhodopsin Photoelectric Transducer: Taking ATP as an Example.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.