Amorphous Aggregation of Amyloid Beta 1-40 Peptide in Confined Space Giulia Foschi, [a] Cristiano Albonetti,* [b] Fabiola Liscio, [c] Silvia Milita, [c] Pierpaolo Greco, [a] and Fabio Biscarini [d] The amorphous aggregation of Ab1-40 peptide is addressed by using micromolding in capillaries. Both the morphology and the size of the aggregates are modulated by changing the contact angle of the sub-micrometric channel walls. Upon de- creasing the hydrophilicity of the channels, the aggregates change their morphology from small aligned drops to discon- tinuous lines, thereby keeping their amorphous structure. Ab1- 40 fibrils are observed at high contact angles. Several neurodegenerative diseases are classified as proteino- pathies as they are associated with the aggregation of misfold- ed proteins. [1–3] Among them, Alzheimer’s (AD), Huntington’s, Parkinson’s and Prion diseases are hallmarked by fibrillar ag- gregates of misfolded amyloidogenic proteins (plaques) be- cause of the overabundant amount accumulated in specific re- gions of the patient’s brain. Amyloid beta (Ab) peptide aggre- gates and accumulates in two types of plaques: diffuse, that is, soluble non-amyloidogenic amorphous aggregates, and com- pact, that is, insoluble amyloidogenic fibrils. [4–5] The insolubility of plaques is one of the neurotoxicity distinctive features, so compact plaques are regarded as neurotoxic whereas diffuse ones are not. [6–7] Ab peptide exhibits two main alloforms, which are 40 (Ab1- 40) and 42 (Ab1-42) aminoacids long, produced by the proteo- lytic cleavage of the amyloid precursor protein (APP) in the cell membrane. [8] In the cerebrospinal fluid (CSF) of normal sub- jects, Ab peptide concentrations are in the nanomolar range. [9] In AD patients, the Ab1-42 concentration decreases by one- third, while the Ab1-40 concentration remains unchanged. [10, 11] Accordingly, Ab1-42 is regarded as a standard AD biomarker, even though Ab1-40 is the dominant peptide species (  90% of the overall Ab peptides in physiological state). [12] The de- crease of Ab1-42 concentration is ascribed to the concurrent formation of insoluble fibrils that accumulate in the brain tissue. The formation of fibrils starts from the self-aggregation pro- cess of transient partially structured conformation in amyloid oligomeric nuclei. [13] Partially structured intermediates, which contain some b-structure, form stable nuclei enriched in b- sheet. Successively, the nucleus evolves into an oligomer that further grows into a fibril, fibril bundles and—at later time— plaques. [14, 15] Alternatively, self-aggregation may lead to the formation of amorphous aggregates of partially structured in- termediates that facilitate nucleation by providing a high local concentration of amyloidogenic intermediates. [16] The self-aggregation process takes place in the extracellular space (ECS), a jelly-like matrix of interconnected channels filled with CSF. [17, 18] Due to the complex shape of cells, the channels (i.e. the gap delimited by cellular membranes) show an uneven width (tortuosity) ranging from 38 to 64 nm, [19] with some dis- tended regions 200–500 nm wide (lakes). [20] Experimental values for gaps and lakes were corrected with diffusion models to  80 and  800 nm, respectively. [21] Such sizes are altered in many pathological and physiological states, so that the CSF flow rate, as well as the molecular flux, is reduced. [22–24] Ab peptides dissolved in the CSF interact chemically with cellular membranes, even though such interactions are com- monly hindered by both chemical heterogeneity of membrane surfaces and molecular crowding. [25, 26] To rationalize them in vitro, surfaces with well-defined physical/chemical properties are to be used as prototype systems for studying the Ab pep- tides adsorption, nucleation, aggregation and fibril formation when dissolved in buffer. [27] Such experiments have been large- ly investigated in bulk solutions where the Ab peptides nuclea- tion has been studied as a function of temperature, pH, shear flow, ionic strength, pressure, as well as in presence of metal ions or small molecules, either experimentally or in silico. [28, 29] In contrast, a limited number of experiments on the self-ag- gregation of peptides in a confined space have been per- formed to date. [30–32] Micromolding in capillaries (MIMICs) and microfluidic systems were used to follow up, in real time, the aggregation of Ab peptides within a confined space. [33–35] In particular, MIMICs has been successfully used for patterning polymers, molecules, and bio-molecules. [36, 37] Selective deposi- tion of solutes in well-defined areas and positions has been achieved by exploiting the capillary imbibition of a liquid solu- tion within micrometric channels. [38, 39] In such an environment, the energy barriers for nucleating stable aggregates of mole- cules or bio-molecules are affected by channel walls and inter- [a] Dr. G. Foschi, Dr. P. Greco Scriba Nanotecnologie S. r. L., Via Corticella 183 - 40128 Bologna (Italy) [b] Dr. C. Albonetti Istituto per lo Studio dei Materiali Nanostrutturati - ISMN Consiglio Nazionale delle Ricerche - CNR Via P. Gobetti 101 - 40129 Bologna (Italy) c.albonetti @bo.ismn.cnr.it [c] Dr. F. Liscio, Dr. S. Milita Istituto di Microelettronica e Microsistemi - IMM, Consiglio Nazionale delle Ricerche - CNR, Via P. Gobetti 101 - 40129 Bologna (Italy) [d] Prof. F. Biscarini Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 183, I-41125 Modena (Italy) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201500602. ChemPhysChem 2015, 16, 3379 – 3384 # 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3379 Communications DOI: 10.1002/cphc.201500602