applied sciences Article The Detection of Long-Chain Bio-Markers Using Atomic Force Microscopy Mark S. Anderson Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Dr., Pasadena, CA 91109, USA; [email protected]; Tel.: +1-818-354-3278 Received: 15 February 2019; Accepted: 22 March 2019; Published: 27 March 2019 Abstract: The detection of long-chain biomolecules on mineral surfaces is presented using an atomic force microscope (AFM). This is achieved by using the AFM’s ability to manipulate molecules and measure forces at the pico-newton scale. We show that a highly characteristic force-distance signal is produced when the AFM tip is used to detach long-chain molecules from a surface. This AFM force spectroscopy method is demonstrated on bio-films, spores, fossils and mineral surfaces. The method works with AFM imaging and correlated tip enhanced infrared spectroscopy. The use of AFM force spectroscopy to detect this class of long chain bio-markers has applications in paleontology, life detection and planetary science. Keywords: long-chain bio-markers 2; AFM 3; tip enhanced spectroscopy 1. Introduction Scanning probe microscopes are fundamental tools in microscopy and nanotechnology. The most widely utilized of the probe microscopes is the atomic force microscope (AFM) that was first described in 1986 by Binnig, Gerber and Quate [1]. With an AFM, a tip mounted on a micro-fabricated cantilever is scanned over the surface and the interaction between the tip and the substrate is detected by monitoring the deflection of the cantilever [2]. These microscopes are remarkable for their ability to image individual atoms or molecules. Related AFM technologies have been developed that broadly include molecular manipulation, force measurement, and tip-mediated spectroscopy. The small size, low power requirements and non-vacuum operation make the AFM a suitable instrument for in situ planetary science missions. Consequently, AFM instruments have been sent to Mars aboard the Phoenix lander mission and an AFM is currently on the Rosetta comet mission [3,4]. The AFM cantilever-stylus mechanism is an inherently sensitive force sensor working down to the pico-newton force range. This force detection capability of the AFM has been harnessed to measure the interaction of the tip with surfaces and individual molecules. The force-distance signal of these AFM tip interactions is known as force spectroscopy (AFM-FS) and provides additional sensing capability to the AFM. This enables the AFM to measure localized hardness, friction and the study of molecular interactions with the AFM tip. In addition, the AFM tip-cantilever mechanism has been used as a localized photo-thermal detector for infrared spectroscopy and to mediate surface enhanced Raman spectroscopy. In this work, the AFM is applied to the detection of long chain molecules on mineral surfaces. Long-chain molecules have been suggested as a class of biomarkers [5–7]. This includes long chain polymers such as intra-cellular polymers (proteins, nucleotides, polysaccharides) and extra-cellular bio-films. Using this rationale, the detection of long chain molecules provides evidence for biotic, fossilized and possible pre-biotic molecules—particularly when used in context with other analytical methods and sample history. Appl. Sci. 2019, 9, 1280; doi:10.3390/app9071280 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 1280 2 of 8 Appl. Sci. 2019, 9, x FOR PEER REVIEW 2 of 8 The basic concept for the detection of long chain molecules using AFM-FS is illustrated in Figure1. The deflectionThe basic of theconcept AFM for cantilever the detection is monitoredof long chain as molecules a function using of AFM distance.-FS is illustrated As the tip in approaches Figure from1. a The long deflection distance, of therethe AFM is no cantilever force (deflection) is monitored on as thea function cantilever. of distance. Once As the the tip tip is approaches in proximity to the surface,from a long there distance, are short there range is no van force der (deflection) Waal attractive on the cantilever. (negative) Once forces the on tip the is in tip proximity and ultimately to the cantileverthe surface, is there positively are short deflected range van when der Waal full attractive contact is(negative) made with forces the on surface. the tip and When ultimately in contact withthe cantilever surface, moleculesis positivelyon deflected the surface when full may con adheretact is made to the with AFM the tip surface. [8]. As When the in tip contact is pulled with away the surface, molecules on the surface may adhere to the AFM tip [8]. As the tip is pulled away from from the surface, molecular interactions produce adhesion forces on the tip until detachment of the the surface, molecular interactions produce adhesion forces on the tip until detachment of the molecule.molecule. The The detachment detachment is is readily readily apparent apparent in the force force-distance-distance plot. plot. The The presence presence of absorbed of absorbed smallsmall molecules, molecules, such such as water,as water, produces produces comparatively comparatively shortshort rangerange forces forces and and abrupt abrupt detachment detachment in the force-distancein the force-distance curve. curve. FigureFigure 1. An 1. An illustration illustration of of how how the the force-displacement force-displacement plot plot can can be beused used to reveal to reveal the pr theesence presence of of long-chainlong-chain molecules. molecules. If long-chainIf long-chain molecules molecules areare present, there there is isa characteristic a characteristic detachment detachment signal signal that canthat becan hundreds be hundreds of nanometersof nanometers away away from from thethe surface depending depending on on the the molecular molecular length. length. A crucialA crucial aspect aspect of of this this work is is that that long long chain chain molecules molecules will produce will produce highly characteristic highly characteristic force- force-distancedistance plots. plots. The The tip tip detachment detachment signal signal is at is atsignificantly significantly greater greater distances distances when when long longchain chain molecules are present on a mineral surface. These mineral surfaces can be planetary rocks, meteorites molecules are present on a mineral surface. These mineral surfaces can be planetary rocks, meteorites or fossils. This approach is an extension of previous work, where AFM-FS has been used to measure or fossils. This approach is an extension of previous work, where AFM-FS has been used to measure the length of purified polymers that have one end chemically tethered to the surface [9]. The AFM- the lengthFS has of also purified been used polymers to detect that the have forces one associated end chemically with the tetheredunraveling to protein the surface polymers [9]. The[10– AFM-FS12]. has alsoForce been spectroscopy used to detect has also the been forces used associated to measure with the the molecular unraveling weight protein distribution polymers of [synthetic10–12]. Force spectroscopypolymers haschemically also been attached used (tethered) to measure to a the surface molecular [13,14]. weight distribution of synthetic polymers chemicallyThe attached use of AFM (tethered) force spectroscopy to a surface [has13, 14been]. expanded in this work to include non-tethered Thebiomolecules use of AFM on mineral force surfaces spectroscopy without has chemical been surface expanded preparation. in this workWhile the to includeprecise molecular non-tethered biomoleculeslength is not on directly mineral measured surfaces without without tethering chemical the surface end of the preparation. molecule to While the surface, the precise the presence molecular lengthof islong not directlychain, high measured molecular without weight tethering molecules the endcan ofbe the readily molecule inferred. to the A surface, molecular the presencelength of distribution can be measured and the lower bound of the polymer length can be determined by the long chain, high molecular weight molecules can be readily inferred. A molecular length distribution detachment length revealed in the force-distance plots. Long-chain polymers may show detachment can be measured and the lower bound of the polymer length can be determined by the detachment signals that are hundreds of nanometers away from the surface depending on the molecular length. lengthIn revealedcontrast, mineral in the force-distance surfaces without plots. such Long-chain organic films polymers are distinct may and show show detachment relatively sharp, signals short that- are hundredsrange ofdetachment nanometers in awaythe force from-distance the surface plots. dependingThis AFM-FS on approach the molecular is demonstrated length. In here contrast, to work mineral surfaceswith without standard such AFM organic instruments films areand distinct tip enhanced and show spectroscopy. relatively sharp,Bio-films, short-range fossils and detachment mineral in the force-distancesurfaces were evaluated plots. This to AFM-FSdemonstrate approach the use isof demonstratedan AFM-FS to discriminate here to work biotic with from standard abiotic AFM instrumentsresidues andon miner tip enhancedal surfaces. spectroscopy. Bio-films, fossils and mineral surfaces were evaluated to demonstrate the use of an AFM-FS to discriminate biotic from abiotic residues on mineral surfaces. 2. Materials and Methods 2. MaterialsThe andexperiments Methods were performed using a Digital Instruments Nanoscope 4 AFM (DI, Santa Barbara, CA, USA) and a Bruker Icon AFM system (Bruker, Santa Barbara, CA, USA). The AFM The experiments were performed using a Digital Instruments Nanoscope 4 AFM (DI, Santa Barbara, CA, USA) and a Bruker Icon AFM system (Bruker, Santa Barbara, CA, USA). The AFM approach and retract sampling rates were 6300 nm/s. The force is a product of the deflection of Appl. Sci. 2019, 9, 1280 3 of 8 Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 of 8 cantilever and its spring constant. Silicon nitride cantilevers were used with spring constants in the rangeapproach 0.14–0.16 and N/m retract (Bruker sampling FESP rates tip, were 225 63 microns,00 nm/s.