# The Detection of Long-Chain Bio-Markers Using Atomic Force Microscopy
## 3 Results
### Biological Samples  
#### 3.2.1 Spores and Algae  
The biological samples tested include spore surfaces and lysed algae samples known to contain long chain organic residues. In order to test a biological surface with a complex set of proteins, a spore surface was first selected for analysis. Spores of _Bacillus atrophaeus_ were deposited on quartz substrates from deionized water suspensions and evaporated under dry nitrogen. The proteinaceous surfaces of these spores were readily probed using AFM-FS. Detailed analysis of these surfaces was previously reported and number of morphogenetic proteins are known to be present in the outer spore coating [18; 19]. For comparison, a lysed algae surface (_Chlorella pyrenoidosis_) was similarly examined using samples that were cast onto microscope slides. Figure 3 shows a comparison of the AFM-FS for the spore surface and algae samples with quartz as a reference. The presence of bio-polymers on the spore surface is clearly revealed in the force plot (Figure 3b) where the detachment distances are detected from 10 nanometers to greater than 100 nanometers away from the surface. The algae sample produces broad, long range force distance signals from detachment of a complex mixture of polymers (Figure 3c). 2.2 Analysis of a Fossil Sample Using Atomic Force Microscope-Force Spectroscopy (AFM-FS) and Tip-Enhanced Infrared Spectroscopy  
A fossil sample was analyzed to illustrate how AFM-FS can be applied to detecting biomarkers in a mineral matrix. The test fossil, described in the experiment section, is a stromatolite that has morphological and chemical complexity formed by interactions between microorganisms, gypsum  
Figure 3: AFM-FS detachment plots comparing a clean mineral surface and complex biological surfaces: (**a**) quartz reference surface; (**b**) spore surface revealing the presence of multiple protein strands; (**c**) a lysed algae surface showing the presence of a complex mixture of polymeric strands. deposition, and gypsum crystal growth. In such structures the evidence for biological origins is apparent due to the presence of microfossils, the spatial distribution of organics, and the stromatolite morphology [15]. Since the AFM-FS mode interrogates a surface at one location at a time, it is useful to select areas that are likely to contain organics. To accomplish this microscopic site selection, the AFM's optical targeting microscope is used to find regions that are amenable to AFM imaging and have morphological interest. In addition, AFM imaging using lateral force or phase contrast imaging can be used to localize soft organic coated surfaces [2]. As additional confirmation for the presence of organic residues, the AFM can be used to remove residues onto the AFM tip for enhanced infrared micro-analysis using methods previously described [20]. The stromatolite fossil was analyzed using successive steps in order to detect organics and bio-polymers: 1. Optical microscopy to target the location for AFM analysis; 2. AFM imaging with friction contrast modes to locate soft areas on the fossil; 3. AFM-FS to detect the presence of long chain polymers; 4. Correlated infrared spectroscopy for detection of organic functional groups removed onto the AFM tip. Using the above targeting approach, Figure 4 shows AFM-FS plot from the surface of the stromatolite fossil. This provides evidence for long-chain polymers remaining in this fossil sample. As further confirmation of the presence of bio-organic residue, Figure 5 shows the infrared spectrum of organic material removed from the fossil surfaces. This was collected onto the AFM tip from the same region that was probed using AFM-FS. The infrared spectrum was acquired from the AFM tip coated with the sample removed from the surface. The infrared spectrum shows functional groups including hydrocarbon, esters, amide I and amide II. The evidence of proteinaceous residue is provided by both the AFM-FS and the amide bands in the infrared spectrum.